OBJECTIVE: Limited data exist regarding knowledge of and compliance to the Centers for Disease Control and Prevention's universal adolescent human immunodeficiency virus (HIV) screening recommendations. Our objective was to assess current guideline knowledge, practice, and perceived barriers to emergency department (ED)-based adolescent HIV screening. METHODS: We administered an anonymous Web-based cross-sectional survey from May 1, 2012, to June 30, 2012, to 1073 physicians from the American Academy of Pediatrics Section on Emergency Medicine LISTSERV. Survey participants were included if they (1) practiced as attending-level physicians, (2) practiced primarily in pediatric emergency medicine or general emergency medicine, and (3) provided clinical care for patients younger than the age of 21 years. The survey examined respondent demographics, knowledge, attitudes, beliefs, practices, and barriers to ED-based HIV screening. Standard descriptive statistics and comparative analyses were performed. RESULTS: A total of 220 responses were obtained; 29 responses were excluded and 191 responses were included in the study. Most of the participants were from urban, free-standing children's hospitals and had an annual ED volume of more than 61,000 patient visits. Respondent knowledge of the Centers for Disease Control and Prevention guidelines was low; less than 40% of the respondents identified correct consent requirements. Only 15.4% of the respondents reported screening for HIV more than 10 times for the prior 6 months. Most frequently cited barriers included concerns for privacy (67.4%), follow-up (67%), and cost-effectiveness (65.4%). Human immunodeficiency virus screening facilitators included availability of health educators (83%), established follow-up (74.7%), and rapid HIV tests (65.2%). CONCLUSIONS: Emergency department clinicians exhibit poor knowledge of adolescent HIV screening recommendations. Current universal screening practices remain low; barriers to screening are numerous. Future efforts should disseminate guideline knowledge, increase rapid HIV testing and health educator availability, as well as reduce adolescent-specific barriers.