The activities of azlocillin, cefotaxime, and amikacin alone and in combination were evaluated in in vitro checkerboard studies, in infected neutropenic mice, and in human volunteers. The combination of cefotaxime plus amikacin was more synergistic in vitro than the others against the Enterobacteriaceae tested, and the combination of azlocillin plus amikacin was more synergistic against Pseudomonas aeruginosa and Staphylococcus aureus. Survival of neutropenic mice infected with Escherichia coli and Klebsiella pneumoniae, respectively, was greater with azlocillin plus amikacin (24 of 40 and 11 of 40) and with cefotaxime plus amikacin (21 of 40 and 17 of 40) than with azlocillin plus cefotaxime (22 of 40 and 3 of 40; P < 0.05). Median serum bactericidal activity in volunteers receiving these antibiotics alone and in combination was -1:8 with most agents and with all combinations tested against 10 strains each of E. coli, K. pneumoniae, P. aeruginosa, and S. aureus. These data suggest that clinical trials with combinations of azlocillin or cefotaxime plus amikacin deserve further study in febrile neutropenic patients.Overwhelming infections with gram-negative bacteria and Staphylococcus aureus occur frequently in granulocytopenic patients (7, 9, 24). Early empiric combination antibiotic therapy is widely used in this clinical situation (7,10,18,24). Most therapeutic regimens include an aminoglycoside, such as gentamicin, tobramycin, or amikacin, and a broad-spectrum beta-lactam, such as carbenicillin or ticarcillin, or a cephalosporin. However, there is need for combinations with even broader activity and less toxicity than the above. The studies recorded in this report on the comparative activities of combinations of amikacin with azlocillin, a new broad-spectrum ureidopenicillin (29), and cefotaxime, a new broad-spectrum cephalosporin (26), were a response to that need. The studies were pursued collaboratively in three laboratories using (i) in vitro susceptibility testing, (ii) infected neutropenic mice, and (iii) serum from human volunteers who had received each drug alone and in combination.