The purpose of this study is to describe and understand prospective science teachers’ knowledge development. This is a longitudinal, multiple case study of four prospective biology teachers’ PCK development during a post-baccalaureate teacher education program. The authors learned that as prospective teachers gained more knowledge and experience, the interaction that develops between teachers’ knowledge of learners and their knowledge of instructional sequences becomes more integrated. In addition, the findings demonstrate a strong relationship exists between science teaching orientations and knowledge of learners and instructional sequences.