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Teachers’ professional knowledge is seen as an important precondition for instructional quality. To date, an information gap exists concerning the influence of chemistry teachers’ content knowledge and pedagogical content knowledge on their teaching. Thus a valid and reliable test instrument was implemented to collect generalizable data in this study’s first stage. To simplify data analysis – especially in large-scale assessments – and to increase test participation willingness, a paper/pencil test using a closed-item format was given to 294 prospective and in service chemistry teachers of different school types, biology and physics teachers, and chemists. Results show significant differences between all samples and the appropriateness of the developed tests. Implications for teacher education concerning several subcategories of professional knowledge could be given.