Eleven povidone-iodine-containing products (Betadine) and chlorhexidine gluconate solution were tested for their ability to inactivate human immunodeficiency virus (HIV) in a cell culture system. All Betadine products completely inactivated the virus at povidone-iodine concentrations of greater than or equal to 0.5% (10- to 20-fold dilutions of stock) except for Betadine Lubricating Antiseptic Gel, which required 2.5% for efficacy (1:2 dilution). Chlorhexidine gluconate completely inactivated HIV at concentrations of greater than or equal to 0.2% (1:100 dilution of laboratory stock; 1:20 dilution of commercial stock). Betadine douche and medicated douche did not inactivate HIV at the concentrations recommended for clinical use (0.33% and 0.25%, respectively) but were effective at povidone-iodine concentrations of 0.5%. Inactivation appeared to be immediate since no difference in efficacy based on length of exposure to the microbicide was detected. Thus, both microbicides are highly effective at killing HIV in vitro.