High-quality after-school programs devoted to science have the potential to enhance students' science knowledge and attitudes, which may impact their decisions about pursuing science-related careers. Because of the unique nature of these informal learning environments, an understanding of the relationships among aspects of students' content knowledge acquisition and attitudes toward science may aid in the development of effective science-related interventions. We investigated the impact of a semester-long after-school intervention utilizing an inquiry-based infectious diseases curriculum (designed for use after-school) on 63 urban students' content knowledge and aspects of their attitudes toward science. Content knowledge increased 24.6% from pretest to posttest. Multiple regression analyses indicated suggested that the "self-directed effort" subscale of the Simpson-Troost Attitude Questionnaire-Revised best predicted increases in students' science content knowledge. The construct "science is fun for me" served as a suppressor effect. These findings suggest that future after-school programs focusing on aspects of attitudes toward science most closely associated with gains in content knowledge might improve students' enthusiasm and academic preparedness for additional science coursework by improving student attitudes toward their perceptions of their self-directed effort.