In conjunction with the Centers for Disease Control and Prevention's (CDC's) national Sentinel Hospital Surveillance System for HIV (human immunodeficiency virus) Infection, we conducted unlinked (anonymous) testing of a systematically selected sample of blood specimens for HIV-1 antibodies to assess the HIV-1 infection level in an urban hospital's catchment population. We excluded specimens from patients with admitting diagnoses often associated with HIV infection. Of the 5,350 specimens tested between April 1988 and October 1989, 106 (2%) were HIV-1 seropositive. HIV-1 seroprevalence in female patients was 1%, and in male patients, 3%. Those 25-44 years of age showed the highest seroprevalence (3.7% in all patients: 6.6% in male patients, 1.1% in female patients). These results demonstrate a high HIV-1 infection level in this patient population and suggest Baltimore hospitals should evaluate the impact and costs of developing routine HIV counseling and testing programs for their patients.