This research developed and evaluated a brief but comprehensive measure of knowledge about sexually transmitted diseases (STDs) for use in research and applied settings. Questionnaire construction involved a review of empirical precedents as well as qualitative work with STD experts (n = 6) and the target population (n = 40). Eighty-five items were piloted (n = 50) and tested (n = 391) with college students. Item- and test-level analyses identified items that were eliminated to shorten the questionnaire. Factor analyses revealed a two-factor model of STD knowledge, including a Cause/Cure factor and a General Knowledge factor. Six supplemental items were added to the final questionnaire for their public health value and resulted in the 27-item STD-Knowledge Questionnaire (STD-KQ). The STD-KQ demonstrated internal consistency (alpha = .86) and test-retest reliability (r = .88) over a brief period. Evidence for the validity of the STD-KQ was obtained through a comparison with a validated HIV knowledge questionnaire (Carey & Schroder, 2002); treatment outcome sensitivity was obtained in response to an educational program. Use of the STD-KQ will enable researchers and health educators to identify knowledge deficits, measure knowledge for theory testing, evaluate risk reduction programs, and assess treatment response in research and applied settings.