INTRODUCTION: Future HIV vaccine efficacy trials with adolescents will need to ensure that participants comprehend study concepts in order to confer true informed assent. A Hepatitis B vaccine trial with adolescents offers valuable opportunity to test youth understanding of vaccine trial requirements in general. METHODS: Youth reviewed a simplified assent form with study investigators and then completed a comprehension questionnaire. Once enrolled, all youth were tested for HIV and confirmed to be HIV-negative. RESULTS: 123 youth completed the questionnaire (mean age=15 years; 63% male; 70% Hispanic). Overall, only 69 (56%) youth answered all six questions correctly. CONCLUSIONS: Youth enrolled in a Hepatitis B vaccine trial demonstrated variable comprehension of the study design and various methodological concepts, such as treatment group masking.