An efficient method for gene replacement in Bacillus megaterium was developed and used to inactivate the chromosomal neutral protease gene (nprM) from strain DSM319. A temperature-dependant suicide vector was constructed to allow replacement of the normal chromosomal copy with an altered version of the nprM gene. One mutant B. megaterium MS941 was selected for further characterization. Measurement of extracellular protease activity from strain MS941 indicated the existence of an additional minor extracellular protease in B. megaterium. Inhibitor studies revealed that this minor protease, comprising only 1.4% of the wild-type total extracellular protease activities, is a serine-type enzyme.