By use of a functional assay for activity, an autolysin structural gene was cloned on a 3 kb EcoRI DNA fragment from a lambda gt11 expression library of Bacillus subtilis 168 trpC2 genomic DNA. Sequencing of the fragment showed five open reading frames, the central one of which encoded the lytic enzyme as found by subclone activity mapping and its homology to a recently sequenced autolysin gene. The protein had a deduced sequence of 272 amino acids and a molecular mass of 29957 Da. When expressed in Escherichia coli DH5 alpha, the protein was processed to a 21 kDa form, as estimated by renaturing SDS-PAGE. The autolysin was an N-acetylmuramyl-L-alanine amidase and its activity was MgCl2-dependent (20 mM optimum) and LiCl-sensitive. The enzyme could bind to and hydrolyse a wide range of peptidoglycan substrates isolated from Gram-positive bacteria; the binding was also MgCl2-dependent. Initial mapping experiments located the autolysin gene near aroD on the B. subtilis 168 chromosome.