What learning opportunities in higher education promote the development of content knowledge (CK), pedagogical content knowledge (PCK), and pedagogical knowledge (PK)? In order to investigate this question, a cross-sectional study with a total of 274 German preservice biology teachers (21.5% male, average age 22.8 years) was conducted in German universities. Preservice teachers were recruited via announcements in teacher education courses. The participation rate amounted to 45%. Results indicate that CK, PCK, and PK are three unique and separable, but correlated domains of knowledge. Regression analyses show how particular learning opportunities are related to preservice biology teachers' CK, PCK, and PK. Both (a) the type of teacher education program and (b) the period of university studies are related to CK and PCK. Moreover, (c) additional subjects studied and (d) teaching experience seem relevant for PCK development. Conclusions for teacher education are drawn.