OBJECTIVE: To assess the level of knowledge regarding and attitudes toward standard and isolation precautions among healthcare workers in a hospital. METHOD: A confidential, self-administered questionnaire survey was conducted in a random sample of 1500 nurses and 500 physicians in a large teaching hospital. RESULTS: A total of 1,241 questionnaires were returned (response rate, 62%). The median age of respondents was 39 years; 71.9% were women and 21.2% had senior staff status. One-fourth had previously participated in specific training regarding transmission precautions for pathogens conducted by the infection control team. More than half (55.9%) gave correct answers to 10 or more of the 13 knowledge-type questions. The following reasons for noncompliance with guidelines were judged as "very important": lack of knowledge (47%); lack of time (42%); forgetfulness (39%); and lack of means (28%). For physicians and healthcare workers in a senior position, lack of time and lack of means were significantly less important (P <.0005). On multivariate linear regression, knowledge was independently associated with exposure to training sessions (coefficient, 0.33; 95% confidence interval, 0.08 to 0.57; P =.009) and less professional experience (coefficient per increasing professional experience, -0.024; 95% confidence interval, -0.035 to -0.012; P <.0005). CONCLUSIONS: Despite a training effort targeting opinion leaders, knowledge of transmission precautions for pathogens remained insufficient. Nevertheless, specific training proved to be the major determinant of "good knowledge".