OBJECTIVE To evaluate the effectiveness of three different types of handcleansing agents in decontaminating gloved hands that were inoculated with a series of four nosocomial pathogens. METHODS A controlled, experimental trial. METHODS Tertiary care referral center. METHODS Five healthy volunteers participated in all portions of the study. METHODS A standard concentration of one of four representative nosocomial pathogens was placed on the gloved hand, spread, and allowed to dry. One of three different handcleansing agents--a nonmedicated soap, a 60% isopropyl alcohol preparation, or 4% chlorhexidine gluconate--was used to cleanse the gloves, which were cultured using a broth-bag technique. The gloves were then removed and the hands were cultured in a similar manner. RESULTS The handwashing agents reduced the median log10 counts of organisms to 2.1 to 3.9 after an inoculation of 10(7) colony forming units. The proportion of positive glove cultures for Staphylococcus aureus, 8% to 100%; Serratia marcescens, 16% to 100%; and Candida albicans, 4% to 60% varied greatly after use of the different handcleansers (P less than 0.001), and varied considerably for Pseudomonas aeruginosa, 20% to 48% (P = 0.085). After the gloves were removed, the differences among the observed proportions of hands contaminated with the test organisms varied from 5% to 50%, depending on the handcleansing agent used (P less than 0.001). CONCLUSIONS In the era of universal precautions these data suggest that it may not be prudent to wash and reuse gloves between patients. Further, handwashing is strongly encouraged after removal of gloves.