One main focus of teacher education research concentrates on teachers' pedagogical content knowledge (PCK). It has been shown that teachers' PCK correlates with teaching effectiveness as well as with students' achievement gains. Teachers' PCK should be analyzed as one of the main important components to evaluate professional development programs. On this account, it is necessary to develop standardized measures of biology teachers' topic-specific PCK that are labor-efficient. This paper presents a study on the development, evaluation, and validation of a paper-and-pencil test to measure biology teachers' declarative PCK on the topic of blood and the human cardiovascular system. The development of the test was based, among other considerations, on a review of research literature on PCK and an analysis of 50 videotaped biology lessons. The final test instrument was comprised of 15 items distributed across 2 scales. The findings of the main study--with 93 preservice and in-service biology teachers and 12 biologists--confirmed that this measure of biology teachers' declarative PCK was reliable, objective, and valid. In-service biology teachers scored higher on the test than preservice teachers (effect size Cohen's d, 0.65) on one hand and, also, than biologists (Cohen's d, 1.00) on the other hand. Future versions of this test should explore enlarging the scales and measuring procedural aspects of PCK.