The effect of chlorhexidine and nonoxynol-9, either singly or in combination, on the replication and infectivity of HIV and the survival of both lymphocytes (MT2 cells) and human spermatozoa, was studied in vitro. Exposure of MT2 cells to 200 microg/ml nonoxynol-9 or 1 mg/ml chlorhexidine for one minute destroyed their viability. A combination of 60 microg/ml of nonoxynol-9 and chlorhexidine, however, killed MT2 cells under the same conditions. Nonoxynol-9 and chlorhexidine were both spermicidal, 268 microg/ml nonoxynol-9, or 2.063 mg/ml chlorhexidine caused complete immobilization of sperm after one minute. The same effect was achieved by a combination of 200 microg/ml nonoxynol-9 and 1.0 mg/ml chlorhexidine. The effect of chlorhexidine and nonoxynol-9 on the replication of HIV was estimated by the output of p24 (the HIV core protein) and the concentration of virus was determined by titration with MT2 cells. Separately, 300 microg/ml nonoxynol-9 alone completely inactivated HIV, while 1 mg/ml chlorhexidine was 80%-100% effective. Certain combinations of nonoxynol-9 and chlorhexidine were antagonistic in their inactivation of HIV, up to 400 microg/ml chlorhexidine partly neutralized the action of 200-500 microg/ml nonoxynol-9.