In a flipped classroom model, learning of basic content is shifted before class while in-class time is used for concept application. Empirical and controlled research studies are lacking on the best strategies to provide the necessary pre-class content instruction. In this study, we tested three methods of pre-class content learning—interactive online tutorials, video lectures, and textbook-style readings—while holding the content and the in-class application activities constant. Identical introductory, non-majors biology classes were manipulated at both a public, open-enrollment institution and a private, highly selective institution. We found that video lectures offer a small advantage to overall student learning over interactive tutorials or textbook-style readings. Although our two populations differed in their ability to effectively learn from pre-class activities, through a student-centered flipped classroom approach, students at both institutions demonstrated equal learning gains by the final assessment. Potential reasons for some observed differences are suggested.