Transmission of human immunodeficiency virus (HIV) in semen by sexual intercourse is believed to be a major etiological factor in the spread of the disease. In order to explore the role of sperm in this transmission, cell-free HIV-l(IIIB) was coincubated with human sperm for 5 h. After incubation, unattached HIV-1 was extensively washed off the cells and the presence and location of HIV-1 in or on the sperm was determined by electron microscopic (EM) and immunofluorescence methods. Examination of sperm at the EM level showed that HIV-1 attaches to the sperm surface and enters the sperm through the intact cell membrane. Immunofluorescence studies, using polyvalent anti-HIV antibodies, demonstrated the presence of small round fluorescent particles in 5–10% of sperm heads after 5 h of incubation. A search for HIV-1 receptors (CD4 epitopes) revealed the presence of OKT4 epitopes, including OKT4A, B, D, and F on 5–10% of sperm by epifluorescence microscopy and on 10–20% of sperm by flow-cytometric analysis. These in vitro data suggest that human sperm may be the primary cellular element in the transmission of HIV by semen.