For assessment of the efficacy of surgical hand disinfection, European reference method prEN 12791 prescribes that the hands must be kept wet with the reference alcohol for 3 min regardless of the applied volume. The aim of this study was to determine whether the applied volume of the reference disinfectant n-propanol (60%, vol/vol) influences the effect on the resident hand flora. Ten experiments with 200 reference disinfections were analyzed. Hands were washed for 1 min with soap. The bacterial prevalue was obtained by rubbing fingertips in tryptic soy broth for 1 min. After this, each subject treated the hands with n-propanol (60%, vol/vol) by using as many portions as necessary to keep hands wet for a total of 3 min. Bacterial postvalues (immediate effect) were obtained for one hand, and the other hand was gloved for 3 h. After the gloves were taken off, a second postvalue was obtained (sustained effect). Most surgical reference disinfections (73%) were achieved with 9 ml of the reference alcohol, followed by 12 ml (24%) and 6 ml (3%). There was no significant difference between the mean log 10 reduction values for the three treatment groups, both in terms of the immediate effect (P ؍ 0.333, as determined by analysis of variance) and in terms of the sustained effect (P ؍ 0.442). A higher number of portions did not correlate with a higher reduction factor (for immediate effect, Pearson's correlation coefficient ؍ ؊0.028 [P ؍ 0.689]; for sustained effect, Pearson's correlation coefficient ؍ 0.059 [P ؍ 0.404]). If the hands were kept wet with the reference alcohol for the total application time, the applied volume could vary, but this did not alter the efficacy.