Infection with the human immunodeficiency virus type 1 (HIV-1), as demonstrated by viral cultures, has been described in some patients before antibodies to HIV-1 can be detected, but the duration and frequency of such latent infections are uncertain. We selected prospectively a cohort of 133 seronegative homosexual men who continued to be involved in high-risk sexual activity, and we cultured 225 samples of their peripheral-blood lymphocytes, using mitogen stimulation to activate the integrated HIV-1 genome. HIV-1 was isolated in blood samples from 31 of the 133 men (23 percent), 27 of whom have remained seronegative for up to 36 months after the positive culture. The other four men seroconverted 11 to 17 months after the isolation of HIV-1. In three of them, we studied cryopreserved lymphocytes obtained earlier, using the polymerase chain reaction to amplify small amounts of viral DNA, and we demonstrated that HIV-1 provirus had been present 23, 35, and 35 months before seroconversion. We conclude that HIV-1 infection in homosexual men at high risk may occur at least 35 months before antibodies to HIV-1 can be detected. A prolonged period of latency in such infections may be more common than previously recognized; the degree of infectiousness during such periods is unknown.