BACKGROUND: Effective neutralization in testing hand hygiene preparations is considered to be a crucial element to ensure validity of the test results, especially with the difficulty to neutralize chlorhexidine gluconate. Aim of the study was to measure the effect of chemical neutralization under practical test conditions according to EN 1500. METHODS: We have investigated two ethanol-based hand rubs (product A, based on 61% ethanol and 1% chlorhexidine gluconate; product B, based on 85% ethanol). The efficacy of products (application of 3 ml for 30 s) was compared to 2-propanol 60% (v/v) (two 3 ml rubs of 30 s each) on hands artificially contaminated with Escherichia coli using a cross-over design with 15 volunteers. Pre-values were obtained by rubbing fingertips for 1 minute in liquid broth. Post-values were determined by sampling immediately after disinfection in liquid broth with and without neutralizers (0.5% lecithin, 4% polysorbate 20). RESULTS: The neutralizers were found to be effective and non-toxic. Without neutralization in the sampling fluid, the reference disinfection reduced the test bacteria by 3.7 log10, product B by 3.3 log10 and product A by 4.8 log10 (P = 0.001; ANOVA). With neutralization the reference disinfection reduced the test bacteria by 3.5 log10, product B by 3.3 log10 and product A by 2.7 log10 (P = 0.011; ANOVA). In comparison to the reference treatment Product B lead to a lower mean reduction than the reference disinfection but the difference was not significant (P > 0.1; Wilcoxon-Wilcox test). Without neutralizing agents in the sampling fluid, product A yielded a significantly higher reduction of test bacteria (4.8; P = 0.02) as compared to the situation with neutralizing agents (2.7; P = 0.033). CONCLUSION: The crucial step of neutralization lies in the sampling fluid itself in order to stop any residual bacteriostatic or bactericidal activity immediately after the application of the preparation, especially with chlorhexidine gluconate-containing preparations. This is particularly important at short application times such as the 30 s.