Muramidase-2 of Enterococcus hirae is a 74-kDa peptidoglycan hydrolase that plays a role in cell wall growth and division. To study its regulation, we isolated a mutant defective in muramidase-2 release under certain growth conditions. This mutant had cell walls which apparently lacked 74-kDa muramidase-2 but which accumulated two proteolytic fragments of 32 and 43 kDa, which exhibited muramidase-2 activity in the membrane fraction. By complementation cloning, we identified a 2.6-kb fragment of the E. hirae chromosome containing a gene cluster coding for proteins of 58 to 137 amino acids. One of these genes (arpU), which encoded a 15.9-kDa protein, was shown to complement the defect of the A9 mutant in trans. We propose that this gene may be involved in the regulation of muramidase-2 export.