Although widely touted as important, there is little evidence regarding the influence of teaching experience on elementary teachers' science subject matter knowledge (SMK). To better understand this phenomenon, we administered an assessment of science topics taught in the fifth and sixth grades to 169 preservice teachers, 231 fifthgrade teachers, and 208 sixth-grade teachers. We then compared the mean scores of teachers at different stages in the career cycle using one-way and two-way ANOVA and explored the relationship between SMK scores and years of teaching experience using regression analysis. Findings indicate that (i) being assigned to a specific grade level had an impact on teachers' SMK for topics included in the grade level, (ii) teachers' SMK scores were lower later in their careers for both science topics they had never taught and for science topics they were responsible for teaching, and (iii) results differed for fifth and sixth grades. This study adds to the existing literature through the examination of a large sample of elementary teachers, with teachers of varying years of experience, while focusing on the science topics these teachers are responsible for teaching. The results of this study provide strong evidence that (i) years of experience teaching specific science topics is associated with the development of teachers' knowledge of these topics, and (ii) teaching experience, what teachers do in the context of their everyday practice, can be an effective means of self-directed learning for teachers. There is also evidence that this influence is not uniform across years of teaching experience or science topics taught. These findings raise important implications about future research into the mechanism of SMK development through teaching experience and teacher grade-level assignments.