OBJECTIVE: To quantify the different behavioral components of healthcare workers' motivation to comply with hand hygiene in a healthcare institution with a 10-year history of hand hygiene campaigning. DESIGN: Cross-sectional study, by use of an anonymous, self-administered questionnaire. SETTING: A 2,200-bed university teaching hospital. PARTICIPANTS: A stratified random sample of 2,961 medical and nursing staff. RESULTS: A total of 1,042 questionnaires (35.2%) were returned. Of the respondents, 271 (26.0%) were physicians, 629 (60.4%) were nurses, and 141 (13.5%) were nursing assistants. Overall, 1,008 respondents provided information about sex; 718 (71.2%) of these were women. Respondents provided demographic information and data about various behavioral, normative, and control beliefs that determined their intentions with respect to performing hand hygiene. Among behavioral beliefs, the perception that healthcare-associated infections are severe for patients was highly ranked as a determinant of behavior by 331 (32.1%) of the respondents, and the perception that hand hygiene is effective at preventing these infections was ranked highly by 891 respondents (86.0%). Among normative beliefs, perceived social pressure from patients to perform hand hygiene was ranked highly by 760 respondents (73.7%), pressure from superiors was ranked highly by 687 (66.8%), pressure from colleagues was ranked highly by 596 (57.9%), and pressure from the person perceived to be most influential was ranked highly by 687 (68.8%). Among control beliefs, the perception that hand hygiene is relatively easy to perform was rated highly by 670 respondents (65.1%). High self-reported rates of adherence to hand hygiene (defined as performance of proper hand hygiene during 80% or more of hand hygiene opportunities) was independently associated with female sex, receipt of training in hand hygiene, participation in a previous hand hygiene campaign, peer pressure from colleagues, perceived good adherence by colleagues, and the perception that hand hygiene is relatively easy to perform. CONCLUSIONS: In a setting with a long tradition of hand hygiene campaigns, behavioral beliefs are strongly in favor of hand hygiene, but adherence is driven by peer pressure and the perception of high self-efficacy, rather than by reasoning about the impact of hand hygiene on patient safety. Female sex, training, and campaign exposure increased the likelihood of compliance with hand hygiene. This additional insight can help to shape future promotional activity.