Combining tazobactam, a beta-lactamase inhibitor, with the ureidopenicillin, piperacillin, successfully restores the activity of piperacillin against beta-lactamase producing bacteria. Thus, piperacillin/tazobactam is highly active against most clinically important species of Gram-negative and Gram-positive bacteria, including anaerobes. We evaluated the in vitro activity of piperacillin/tazobactam against clinical isolates from a tertiary university hospital located in Sao Paulo, Brazil. Its activity was compared to that of ticarcillin/clavulanic acid, ampicillin/sulbactam, ceftazidime, ceftriaxone, cefotaxime, cefoxitin, aztreonam, and imipenem against 820 isolates (608 Gram-negative and 212 Gram-positive) collected from hospitalized patients in 1999. The most frequent species tested were Pseudomonas aeruginosa (168/20%), Escherichia coli (139/17%), Acinetobacter spp. (131/16%), and Staphylococcus aureus (76/9%). Of the isolates studied, 30% were from the bloodstream, 16% from the lower respiratory tract, and 11% from surgical wounds or soft tissue. The isolates were susceptibility tested by the broth microdilution method according to NCCLS procedures. The isolates tested were highly resistant to most antimicrobial agents evaluated. Imipenem resistance was not verified among Enterobacteriaceae, and piperacillin/tazobactam was the second most active beta-lactams against this group of bacteria (80.0% susceptibility). Extended-spectrum beta-lactamase production was very high among E. coli (approximately 20%) and Klebsiella pneumoniae (approximately 40%). Imipenem was uniformly active against these species (100% susceptibility) and piperacillin/tazobactam was the second most active compound inhibiting 84.4% of isolates. Pseudomonas aeruginosa was highly resistant to all beta-lactams evaluated and piperacillin/tazobactam was the most active compound against this species. Our results demonstrate an extremely high level of antimicrobial resistance in the hospital evaluated, especially among non-enteric Gram-negative bacilli. Due to this high level of resistance, piperacillin/tazobactam represents an important contribution to the treatment of nosocomial infections.