OBJECTIVES: Evaluation of the impact of the implementation of practice guidelines, with or without their reinforcement by a pharmacist, on the intra-hospital use of antibiotics. MATERIALS AND METHODS: The duration of antibiotic treatment, their cost, and the length of patient stay were compared in three secondary-care hospitals, before and after interventions that were designed to promote rational antibiotic use. After randomization, hospital A received no intervention (control), local practice guidelines were implemented in hospital B (low grade intervention), and these guidelines were reinforced by a clinical pharmacist in hospital C (high grade intervention). Adherence to the guidelines was measured in hospitals B and C. Multivariable statistical analyses were carried out to adjust for confounding factors. RESULTS: None of the outcomes measured in the 1200 included patients decreased between the two study periods in any hospital. Hospital A was significantly and independently associated with an increase in the duration of antibiotic treatments, the cost of antibiotics (acquisition and global costs), and the length of stay. Although these differences were not statistically significant, increases in hospital B were higher than in hospital C. Adherence to guidelines was significantly higher in hospital C. CONCLUSIONS: Even though interdisciplinary interventions aiming at rationalizing antibiotic use could not diminish the duration of treatments, their costs or the length of stay, they proved useful to control the progression of these parameters.