Objective: The objective of this study was to determine the acceptability of urine screening for Neisseria gonorrhoeae and Chlamydia trachomatis in adolescents in a pediatric emergency department. Study Design: We used a prospective enrollment of adolescents aged 14-20 visiting an urban pediatric emergency department. Main Outcome Measures: The main outcome measure was acceptance of urine STD screening rates. Results: Of 1231 potential participants, 879 (71%) agreed to participate and 352 (29%) declined screening. Participants were similar to those refusing to participate in terms of gender. In multivariate analysis, age, race/ethnicity, and insurance status were associated with variation in sexually transmitted disease (STD) test acceptance, whereas the presence of a parent was not. Despite similar training, 1 of 3 recruiters had significantly lower acceptance rates than her peers. Overall, 10% of patients enrolled were found to have one or both infections. Conclusion: Urine screening for STDs can be efficiently conducted in an emergency department setting. This screening appears to be acceptable to most patients.