Cefazaflur was tested in vitro against 262 strains of bacteria. Inhibitory and bactericidal concentrations were determined with two inoculum sizes of bacterial cells in Mueller-Hinton broth and nutrient broth. Agar dilution studies also were performed. When tested in agar, 5.0 mug or less of cefazaflur per ml inhibited almost all strains of Staphylococcus aureus, Escherichia coli, Klebsiella, and Proteus mirabilis. The drug was less active against Enterobacter and indole-positive Proteus, and 7.5 mug of antibiotic per ml inhibited approximately two-thirds to one-fourth of the strains. A concentration of 50 mug of cefazaflur per ml was required for inhibition of the enterococci. There was negligible activity against Pseudomonas. The drug demonstrated less activity in broth than in agar, and a major inoculum effect was seen with some strains. For example, with a lower inoculum, 2.5 mug of cefazaflur per ml killed all strains of E. coli, whereas with the higher inoculum, 7.5 mug of cefazaflur per ml, inhibited 64% and killed only 8% of strains. The activity of the drug for some strains was greater in Mueller-Hinton broth; for others, it was greater in nutrient broth. There were considerable differences in the results of the broth and agar studies for some species when the same medium was employed. Because of differences in activity found with different media, inocula, and method of testing, an evaluation of the eventual usefulness of cefazaflur must await the results of in vivo studies.