The Centers for Disease Control and Prevention has published a new guideline on hand hygiene promoting the use of the alcohol-based handrub, but the technique was not addressed. The goal of this study was to evaluate the influence of technique on the efficacy of the alcohol-based handrub. Healthcare workers (HCWs) attending a course in hospital epidemiology. A fluorescent dye was added to a hand antiseptic, and hands were checked under ultraviolet light after antiseptic cleansing. Data regarding the numbers of predefined fluorescent areas on the skin were collected in addition to demographic data such as age, gender, job description, and job experience. Results of the visualization test were compared with the data from microbiological samples before and after the procedure by the hand plate technique. Sixty HCWs were tested, 63% of whom had worked in infection control for more than 10 years. Sixty-six percent of all participants still had detectable bacteria after antisepsis. The mean log10 CFU reduction was 2.0 (range, 0-3.85). Twenty-five percent of all HCWs achieved less than 1.1 log10 CFU. Staphylococcus aureus was isolated from 13% (one of them being methicillin resistant) and gram-negative bacilli from 6.7%. After using the alcohol handrub, one subject still remained positive for S. aureus. Years of experience was the single most important factor predicting antimicrobial efficacy. Technique is of crucial importance in hand antisepsis. Major deficiencies were detected among even highly trained HCWs. Training should be provided before switching from handwashing to the alcohol handrub