The first major trial in which the ACLU participated was the Scopes "Monkey Trial." On 21 March 1925, Austin Peay, the governor of Tennessee, signed into law the Butler Act, which made it illegal for any teacher in a state-supported school to teach that humans are descended from a lower order of animals. The author of the bill, J. Washington Butler, was a fundamentalist who feared the impact of science on religion. At issue were several overlapping concerns: academic freedom, freedom of religious expression, governmental authority, and parental rights. Lucille Milner of the ACLU spotted the case in a Tennessee newspaper and brought the issue to Baldwin’s attention. According to Milner, he "saw its import in a flash" and decided to inform the board. The board agreed to enter the controversy and placed an announcement in the Tennessee newspapers offering services to any teacher who would agree to challenge the law. George W. Rappleyea, a young engineer, read of the Union’s offer in the 4 May 1925 edition of the Chattanooga Times and quickly sought a client fdr the ACLU; John T. Scopes, a high school teacher, agreed to challenge the law.

Although the trial was billed as "God Against Monkey" or religion versus science, the recent work of historian R. M. Cornelius indicates that the trial began as a public relations scheme to attract attention to the depressed economic conditions in Dayton, Tennessee. That may have been the motive of Rappleyea, but it was not Baldwin’s. In any event, the trial became a showdown between William Jennings Bryan, prosecutor and three-time presidential candidate, and Clarence Darrow, the noted criminal lawyer. Dudley Field Malone and ACLU attorney Arthur Garfield Hays assisted in the defense, and the Union marshaled the input of Felix Frankfurter, Norman Thomas, Elizabeth Gurley Flynn, Father John Augustine Ryan, and, of course, Baldwin. As Cornelius has observed, the trial became a “tour de farce” as eccentrics of every kind appeared: Joe Mendi - a trained chimpanzee - was there along with Deck "Bible Champion of the World" Carter and Louis Levi Johnson Marshall, "Absolute Ruler of the Entire World, Without Military, Naval or Other Physical Force." Throngs of journalists showed up, including H. L. Mencken, Joseph Wood Krutch, and Westbrook Pegler.

Cornelius wrote, "A stranger trial there probably never was." Members of the jury were caught up more in the drama of the event than in the proceedings themselves. Former colleagues and acquaintances were now adversaries; both Darrow and Malone had assisted the political ambitions of Bryan. Scopes had been in the graduating class at Salem High School when Bryan delivered the commencement address. Scientific experts came from all over to testify; but none of their statements was allowed as evidence. Darrow was cited for contempt and Bryan took the witness stand. Scopes was never called to testify. Moreover, Scopes was a math teacher who only periodically taught biology as a substitute. He later admitted, "To tell the truth, I wasn’t sure I had taught evolution." No matter. If Scopes was found guilty (his defense counsel did not claim he was innocent but asked the jury to find him not guilty), Bryan would pay his fine, contending that the law should not have had a penalty.

The outcome is well known. Scopes was found guilty and fined $100. When the ACLU appealed the case, the Tennessee Supreme Court upheld the law but reversed the conviction. The fine had been imposed by the presiding judge instead of by the jury, as it should have been. The intended effect was obvious: there could be no appeal to the U.S. Supreme Court because the lower court ruling had been reversed on a technicality.