The aim of the study was to develop a sensitive culture method for the detection of HIV in semen. Antimicrobials were evaluated to suppress bacterial and fungal contamination of HIV cultures. Toxicity of seminal plasma was reduced using a short incubation of seminal fluid with culture cells. The detection rate was determined by adding known amounts of diluted primary virus isolates to uninfected cells. The sensitivity of the method was determined in a cohort of 33 HIV seropositive men. The use of a virus transport medium with high doses of antibiotics reduced contamination of HIV cultures. Toxicity of seminal plasma on culture cells was limited when seminal fluid was incubated with culture cells for only 90 min. Detection levels for cell-associated virus and cell-free virus from semen were 12 infected cells per ejaculate and 100 infectious units per milliliter of seminal fluid, respectively. Cell-associated HIV could be recovered from 18 semen samples of 33 HIV positive men (55%). Recovery of cell-free virus from seminal fluid was infrequent. It is concluded that the method is highly sensitive for the detection of HIV from semen. However, the recovery rate of infectious cell-free virus in seminal fluid from clinical samples is infrequent.