OBJECTIVE To determine the diagnostic accuracy of an investigational test for human immunodeficiency virus (HIV) envelope antibodies in urine. METHODS Matched blood and urine specimens were tested for HIV by two independent laboratories, both of which were blinded to all results at the other site. Duplicate positive enzyme-linked immunoassay (EIA) results were confirmed by immunofluorescent antibody or western blot. METHODS Six alcohol treatment centers in the San Francisco metropolitan area. METHODS Five hundred ninety-two recovering alcoholics. METHODS Diagnosis of HIV infection by blood and urine EIA and western blot. RESULTS The experimental urine EIA, when confirmed by urine western blot, led to a correct diagnosis in all samples. One sample was negative by urine EIA screening, positive by blood EIA, and exhibited an indeterminate blood western blot pattern (p24 band only). CONCLUSIONS We encountered no false positive or false negative results using an investigational HIV antibody test for urine samples. There are several important advantages to HIV testing of urine versus serum or blood; however, there are also cogent reasons for limiting the use of alternative specimens for HIV testing.