The association between emergency department (ED) characteristics, ED director's perceptions of preventive services, and the availability of human immunodeficiency virus (HIV) screening are unknown. The authors hypothesized that, after adjusting for ED operational and demographic characteristics, teaching hospital status would be associated with increased availability, and ED crowding and ED director agreement with barriers to screening would be associated with decreased availability. This was a secondary, cross-sectional analysis on previously collected data from 2008 to 2009 regarding availability of ED preventive services. Data were obtained from a random sample of 277 EDs in which ED directors provided information on ED characteristics and availability of HIV screening and rated five barriers to providing preventive services. The association between the availability of HIV screening and teaching hospital and crowding status, ED volume, urban-rural location, ownership, geographic region, patient demographics, state HIV testing consent laws, and ED director opinions on barriers to providing preventive services were determined in univariate analyses and a multivariate logistic regression model. Nineteen percent of the sampled EDs offer HIV screening. Teaching hospitals offer HIV screening more frequently than nonteaching hospitals (38% vs. 18%; p = 0.03), but after adjusting for other characteristics in a multivariate model, this association was not significant (relative risk ratio [RR] = 2.07, 95% confidence interval [CI] = 0.91 to 3.59). ED crowding also was not significantly associated with screening availability (RR = 0.66, 95% CI = 0.34 to 1.21). However, public ownership (RR = 2.13, 95% CI = 1.28 to 3.14), 24-hour social work (RR = 1.87, 95% CI = 1.02 to 2.99), uninsured population ≥35% (RR = 2.48, 95% CI = 1.39 to 3.69), increased local nonwhite minority population percentage (RR = 1.14 per 10%, 95% CI = 1.02 to 1.26), and state laws allowing opt-out consent for testing (RR = 1.76, 95% CI = 1.01 to 2.74) were associated with increased availability of screening in multivariable analysis. EDs whose directors were concerned about added costs were associated with decreased availability of screening (RR = 0.45, 95% CI = 0.23 to 0.85). After adjusting for other ED operational and demographic characteristics, ED crowding and teaching hospital affiliation were not independently associated with the availability of HIV screening. EDs whose directors were concerned about the cost of preventive services were less likely to provide routine HIV screening. Addressing ED director's concerns about the added costs of ED preventive services, increasing social work availability, and implementing testing laws consistent with Centers for Disease Control and Prevention (CDC) recommendations may facilitate increased adoption of ED HIV screening.