Use of antimicrobial drugs is associated with the emergence of resistant bacteria, increasing hospital expenditures and requires an assessment of appropriateness. The aim of this study was to investigate prevalence and patterns of antibiotic prescribing and expenditures in Italian hospitals. A prevalence survey was performed in 15 Italian hospitals. Data on antimicrobial use and expenditures in the year 2000 and 1-day point prevalence information on antibiotic prescription, type and sources of infections and expenditures were obtained through a review of pharmacy records and charts of 2,165 inpatients. 45.5% of the patients surveyed were receiving at least one antibiotic: 148 for hospital-acquired infections (HAI), 262 for community-acquired infections (CAI). No active infection was recorded for 575 patients (58.4%). 57.3% of prescriptions for HAI and 65.9% for CAI were based on empiric and presumptive criteria, while 39% of HAI and 15.8% of CAI were oriented by microbiology and susceptibility testing results. Guidelines were followed in choosing the antimicrobial drugs for 22.8% of therapeutic and 46.8% of prophylactic courses. High rates of antibiotic usage were observed in Italian hospitals. Areas for improvement, both for the use of diagnostic resources and the prescribing habits, were identified. The 1-day prevalence survey of antimicrobial use may be a tool of feedback to prescribers for more appropriate drug selection.