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The play opens with the town's excitement over the upcoming trial of one of its schoolteachers, Bert Cates. In his classroom, Cates dared to instruct his students in Darwin's theory of evolution, violating a state law that prohibits the teaching of evolution. A well-known defense lawyer has agreed to take Cates' case. An even more famous and popular attorney and politician, Matthew Harrison Brady, has agreed to be the lawyer for the prosecution. When Brady arrives, he is greeted with a great deal of fanfare. In response to his welcome, he gives a speech that is greatly praised; he is then honored at a picnic. The town's minister, Reverend Brown, welcomes Brady warmly, and Hillsboro's mayor presents him with an award as an honorary colonel of the state militia.
At the courthouse, Rachel Brown visits the defendant, Bert Cates. She is Cates' girlfriend, a fellow teacher in his school, and the daughter of the town's minister. Rachel begs Bert to say he made a mistake in presenting evolution to his class in order to stop the trial from beginning; she is fearful of what will happen to Cates during the trial. E.K. Hornbeck, a reporter form the Baltimore Herald, arrives at the courthouse to see Cates. He praises the defendant for being so brave. Rachel does not think that Cates deserves to be praised, for he broke the law and opposed the teachings of the Bible.
In the courtroom a few days later, the selection of the jury begins. Brady looks for people who are believers in the Bible and who think that evolutionary theory is evil. In contrast, Drummond looks for people who have not formed opinions about evolutionary theory and who are not overly religious. Drummond objects to the fact that Brady is being called Colonel Brady by the judge; he claims it gives his opponent an unfair advantage. The mayor is brought in to resolve the issue. He also grants Drummond the title of "Colonel" on a temporary, honorary basis. Now both lawyers are called Colonel.