A cross-sectional study was conducted to evaluate the utility of a commercial enzyme immunoassay (EIA) as a screening test for detecting HIV-1 antibody in urine in a population at risk for HIV infection in Catalonia, Spain. Paired urine and serum samples were collected consecutively from 99 patients who attended two drug-dependency treatment centres and 151 patients who attended a sexually transmitted diseases (STD) clinic in Barcelona. Antibodies against HIV in urine samples were detected using the Calypte HIV-1 Urine EIA (Calypte Biomedical Corporation, Berkeley, CA, USA) and confirmed by urine-based Western blot (WB) analysis. Sera were analysed using Bioelisa HIV-1+2 EIA (Biokit Laboratories, Barcelona, Spain), and the results were verified using serum-based WB analysis. Results of both urine and serum testing were available for 246 of 250 participants. For 52 individuals the results of both urine and serum testing were positive and for five the results were discordant (2 with urine-negative/serum-positive results and 3 with urine-positive/serum-negative results). The respective sensitivity and specificity values obtained for the urine EIA were 100% and 96.2% for intravenous drug users (IDUs) and 80% and 99.3% for persons attending the STD clinic. According to the 1997 UNAIDS/WHO strategy I recommendations, these values are acceptable for surveillance purposes, particularly in populations with a high prevalence of HIV infection.