The effect of cerulenin on the production of fi-lactamaseand other periplasmic proteins was studied in Escherichia coli IA199 carrying plasmid pBR322. Cerulenin (10 to 25 #g/ml) had almost no effect on the growth rate of E. coil but it decreased the amount of fl-lactamase and other periplamic proteins in shock fluid. Higher amounts of the antibiotic (40 to 100 #g/ml)decreased turbidity and almost completely prevented synthesis offl-lactamase and other periplasmic proteins. Cerulenin decreased incorporation of L-[BSS]methionine into membranes during growth as well. Spheroplasts secreted fl-lactamase into the external medium, but during a 3-h incubation in the presence of cerulenin (25 #g/ml) this secretion was prevented by more than 90%. fi-Lactamase was secreted into the isolated membrane vesicles from E. coli IA199. However, only 5~ of the total amount of pre-fi-lactamase was secreted and processed by the membranes in vitro. Cerulenin did not prevent processing & vitro but the membranes prepared from the cells grown in the presence of cerulenin (25 #g/ml) did not catalyze processing of pre-fi-lactamase at all. Membrane preparations from Bacillus subtilis did not process pre-fi-lactamase either in the absence or in the presence of cerulenin.