The period of latency between infection by the human immunodeficiency virus type-1 (HIV-1) and the production of specific antibodies to viral antigens may be prolonged and, occasionally, may last for years. This condition of seronegative infection could represent a serious risk of viral transmission from subjects who are unaware of their status. However, whether these individuals are actually infectious, especially through body fluids, has not been clarified. We have performed a prospective study in 65 high-risk individuals seronegative for HIV-1 antibodies for a prolonged period of time. Twelve of them (18%) were shown to be carriers of HIV-1 proviral sequences by the polymerase chain reaction (PCR). The virus was isolated from mitogen-stimulated peripheral blood lymphocytes in five out of ten subjects tested since the first positive PCR. In two of them, virus could also be isolated from cell-free plasma, subsequently they remained seronegative during 10 months of follow-up. These data indicate that delayed seroconversions may be associated with productive infection, suggesting that mechanism(s) other than viral latency may be responsible for the absence of antibody responses to HIV-1 proteins. Furthermore, our findings suggest that prolonged seronegative individuals can transmit HIV infection through their body fluids.