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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.We used a prospective enrollment of adolescents aged 14–20 visiting an urban pediatric emergency department.The main outcome measure was acceptance of urine STD screening rates.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.Urine screening for STDs can be efficiently conducted in an emergency department setting. This screening appears to be acceptable to most patients.