The in vitro activity of cefoxitin, 3-carbamolyloxymethyl-7-α-methoxy-7[2-(2-thienyl)acetamido]-3-cephem-4-carboyxlic acid, was investigated. Activity against gram-positive organisms was less than that of cephalothin and cephloridine. It was highly active against gram-negative bacilli, with activity against Escherichia coli, Proteus mirabilis, and Klebsiella pneumoniae equal to that of currently available cephalosporins. In addition, it was active against certain Enterobacter strains, Serratia marcescens, indole-positive Proteae and Herellea. The strains of these latter bacteria were strains susceptible to carbenicillin and ticarcillin. Pseudomonas aeruginosa and other Pseudomonas species were resistant. Changes in pH, inoculum size, and type of growth medium had no significant effect on the activity of the antibiotic. Cefoxitin was highly resistant to hydrolysis by various types of gram-negative beta-lactamases. The precise role of resistance to beta-lactamase hydrolysis varied from strain to strain. Bacterial resistance to cefoxitin was not necessarily related to hydrolysis of the antibiotic. However, the resistance of cefoxitin to hydrolysis did contribute to its activity. Cefoxitin could function as an inducer of beta-lactamase activity and effectively bound to purified beta-lactamases.