This report explores the level of detailed knowledge about the Tuskegee Syphilis Study (TSS) among 848 Blacks and Whites in three U.S. cities across an array of demographic variables. The Tuskegee Legacy Project (TLP) Questionnaire was used, which was designed to explore the willingness of minorities to participate in biomedical studies. A component of the TLP Questionnaire, the TSS Facts & Myths Quiz, consisting of seven yes/no factual questions, was used to establish respondents’ level of detailed knowledge on the TSS. Both Blacks and Whites had similar very low mean quiz score on the 7-point scale, with Blacks’ scores being slightly higher than Whites (1.2 vs. 0.9, p = .003). When analyzing the level of knowledge between racial groups by various demographic variables, several patterns emerged: (a) higher education levels were associated with higher levels of detailed knowledge and (b) for both Blacks and Whites, 30 to 59 years old knew the most about TSS compared with younger and older adult age groups. The findings show that much of the information that circulates in the Black and White communities about the TSS is false, often minimizing or understating the most egregious injustices that occurred. Health promotion and educational implications of these findings are offered and conclude that the findings should be used as a catalyst to explore local realities and sentiments regarding participation in biomedical research within the research philosophy and framework of community-based participatory research.