Piperacillin was evaluated in vitro against 711 clinical isolates of aerobic and anerobic gram-positive and gram-negative bacteria, including 76 isolates of Salmonella typhi. Piperacillin minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were compared with those of a range of β-lactam, aminoglycoside, and other antimicrobial agents, and inoculum size effects were considered. The relationship between dilution and disk diffusion tests was studied by regression analysis. In addition, piperacillin was assessed in combination with aminoglycoside and other β-lactam drugs. This investigation has confirmed the activity of piperacillin against a broad range of bacteria, including Pseudomonas, Enterobacteriaceae, Neisseria, β-lactamase-negative Haemophilus influenzae, and Staphylococcus aureus as well as enterococci, Bacteroides fragilis, and other anaerobes. All strains of Pseudomonas aeruginosa were inhibited by ≤32 μg/ml or less, demonstrating again the potential usefulness of piperacillin in the treatment of pseudomonal infections. S. typhi proved susceptible to piperacillin, all isolates being inhibited by 1 μg/ml. Inoculum size experiments showed that inocula of 108 CFU resulted in MICs and MBCs appreciably higher than those resulting from inocula of 106 CFU, and inocula of 102 CFU resulted in MICs and MBCs appreciably lower than those resulting from inocula of 104 CFU. Piperacillin was active against all gentamicin-resistant pseudomonads tested, but not against gentamicin-resistant klebsiellas and enterobacters. Combinations of piperacillin with tobramycin and amikacin were consistently synergistic against Pseudomonas and Serratia isolates. Less consistent results were shown when piperacillin was combined with aminoglycosides or cephalothin against Klebsiella and indole-positive Proteus isolates, although synergy was observed in most cases. Occasional antagonistic reactions were encountered with piperacillin-cephalothin or piperacillin-tobramycin combinations against the latter isolates.