The literature on hand washing, while extensive, often contains conflicting data, and key variables are only superficially studied or not studied at all. Some hand washing recommendations are made without scientific support, and agreement between recommendations is limited. The influence of key variables such as soap volume, lather time, water temperature, and product formulation on hand washing efficacy was investigated in the present study. Baseline conditions were 1 mL of a bland (nonantimicrobial) soap, a 5-s lather time, and 38°C (100°F) water temperature. A nonpathogenic strain of Escherichia coli (ATCC 11229) was the challenge microorganism. Twenty volunteers (10 men and 10 women) participated in the study, and each test condition had 20 replicates. An antimicrobial soap formulation (1% chloroxylenol) was not significantly more effective than the bland soap for removing E. coli under a variety of test conditions. Overall, the mean reduction was 1.94 log CFU (range, 1.83 to 2.10 log CFU) with the antimicrobial soap and 2.22 log CFU (range, 1.91 to 2.54 log CFU) with the bland soap. Overall, lather time significantly influenced efficacy in one scenario, in which a 0.5-log greater reduction was observed after 20 s with bland soap compared with the baseline wash (P = 0.020). Water temperature as high as 38°C (100°F) and as low as 15°C (60°F) did not have a significant effect on the reduction of bacteria during hand washing; however, the energy usage differed between these temperatures. No significant differences were observed in mean log reductions experienced by men and women (both 2.08 log CFU; P = 0.988). A large part of the variability in the data was associated with the behaviors of the volunteers. Understanding what behaviors and human factors most influence hand washing may help researchers find techniques to optimize the effectiveness of hand washing.