Data on the concentrations of didanosine (ddI) and tenofovir (TFV) in seminal plasma are sparse. Subtherapeutic drug concentrations within the lumen of the male genital tract may have implications for selection and transmission of drug-resistant HIV strains. On the other hand, sufficient penetration of these drugs into the male genital tract has potential toxic effects on the spermatozoa and their precursors. In the current study, the authors obtained paired semen and blood samples at variable time points after drug intake from 30 HIV-1-infected patients using a ddI (n = 15) or ddI + TFV (n = 15) containing an antiretroviral regimen. Didanosine and TFV concentrations were measured in seminal and blood plasma and semen quality was assessed. Both ddI and TFV penetrated well into seminal plasma. Whereas blood plasma ddI concentrations dropped to near or below the lower limit of quantification of 0.017 microg/mL 9 hours after drug intake, the ddI concentration in seminal plasma remained detectable during the whole dosing interval with a median of 0.20 and 0.21 microg/mL in the ddI and ddI + TFV groups, respectively. Tenofovir was detectable during the whole dosing interval in both blood and seminal plasma with a median concentration of 0.12 and 0.25 microg/mL, respectively, and a median seminal-to-blood-plasma ratio of 3.3. Semen quality was within the normal range according to the criteria of the World Health Organization, except for the percentage of progressively motile sperm, which was low in both groups of patients. The authors conclude that ddI and TFV penetrate well into seminal plasma and that the reduced sperm motility deserves further study.