Seventy-five strains of nine species of gram-negative organisms were tested for susceptibility to cephapirin, a new cephalosporin antibiotic, and 23% were found to be resistant. Two of the resistant organisms, an Escherichia coli and a Serratia marcescens, consistently produced cephalosporinase in their filtrates. The E. coli filtrates produced by 109 organisms per milliliter were much more active than those produced by an equal number of S. marcescens. The activity of the S. marcescens filtrate was destroyed by heating at 65 °C for 15 min while that of the E. coli filtrate required heating at 100 °C for 15 min for inactivation. The molecular weight of the filtrate cephalosporinase was estimated to be 10–30 000 daltons by membrane-filtration techniques. The Km for cephapirin inactivation by the cephalosporinase of the S. marcescens filtrate, at its pH optimum of 7, was 49 nanomolar. The extracellular cephalosporinases were active against cephapirin, cephalothin, cephaioridine, and penicillin G at the same dilutions of the filtrates.