The role of herpes simplex virus (HSV) in male infertility has been investigated using the localizing ability of in situ hybridization technique. Sperm samples obtained from 80 men attending a maternity center because of fertility problems were classified by spermogram and analyzed for the presence of HSV-1 and HSV-2 DNA using digoxigenin-labelled DNA probes. HSV DNA was detected in the nuclei of spermatozoa in 37 semen samples (46%), HSV-1 specifically in 21 cases (26%) and HSV-2 in 16 cases (20%). HSV infection was almost three times more common in semens with a sperm count lower than 20 million/ml in relation to semens with a sperm count higher than 20 million/ml (70% versus 25.5%, P = 0.0001). The mean sperm count and motility for HSV positive semens were 23.5% and 36% respectively, whereas those of HSV negative semens were 53.2% and 47% respectively (P(count) = 0.0005 and P(motility) = 0.01). The number of HSV positive sperm cells ranged from 2 cells per specimen to 60% of the sperm cells. The mean number of HSV-2 labelled sperm cells per sample was 3.7% and that of HSV-1 1.5%. The percentage of hybridization positive sperm cells was also inversely correlated with sperm count and motility. Acyclovir therapy of eight males with HSV positive semens resulted in three successful pregnancies. In conclusion, HSV seems to play an important role in male infertility and the detection of this virus in the semen will not only allow substantial therapeutic interventions for the restoration of fertility but it will also contribute to the control of the transmission of HSV infection.