The effects of combining the new broad-spectrum penicillins piperacillin and mezlocillin with cefoxitin, cefamandole, or cephalothin on the antibacterial activities of these antibiotics were determined in vitro against 50 to 109 bacterial strains and in six experimental infections in mice. Against strains of Escherichia coli, Klebsiella, Proteus mirabilis, Salmonella, Acinetobacter, Enterococcus, and Staphylococcus, the combinations exhibited synergistic, indifferent, or additive effects, but no antagonism. Against strains of four groups of organisms (Pseudomonas, Serratia, Enterobacter, and indole-positive Proteus), a high incidence of antagonism was observed, particularly with combinations containing cefoxitin (60 to 100%). The penicillins were antagonized by the cephalosporin antibiotics. In vitro effects were reflected in vivo. Mice infected with cultures associated with synergistic or additive in vitro effects were protected with lower doses of piperacillin when this antibiotic was administered with ineffective doses of cefoxitin than when piperacillin was used alone. Infections with cultures associated with in vitro antagonism required two-to eightfold higher doses of piperacillin and mezlocillin when these antibiotics were used in combination with the cephalosporins. The clinical implications of these effects should be considered.There is considerable information on the synergistic actions of combinations of semisynthetic penicillins or cephalosporins and aminoglycoside antibiotics, but data on the interactions between penicillins and cephalosporins are more limited. Recently, both synergistic and antagonistic effects have been reported for f8-lactam antibiotic combinations (1-6, 8, 9). To obtain a broader understanding of this problem, we studied the in vitro effects of combinations of the new broadspectrum semisynthetic penicillins piperacillin and mezlocillin with the cephalosporins cephalothin, cefamandole, and cefoxitin against bacterial strains belonging to 10 genera. In addition, we tested several combinations for activity against experimental infections in mice.