Cosmid B577, a member of the collection of ordered clones corresponding to the genome of Mycobacterium leprae, contains a gene, provisionally called pon1, that encodes an 821-amino-acid-residue high-molecular-mass class A penicillin-binding protein, provisionally called PBP1. With similar amino acid sequences and modular designs, M. leprae PBP1 is related to Escherichia coli PBP1a and PBP1b, bienzymatic proteins with transglycosylase and transpeptidase activities. When produced in E. coli, His tag-labelled derivatives of M. leprae PBP1 adopt the correct membrane topology, with the bulk of the polypeptide chain on the surface of the plasma membrane. They defy attempts at solubilization with all the detergents tested except cetyltrimethylammonium bromide. The solubilized PBP1 derivatives can be purified by affinity chromatography on Ni2+-nitrilotriacetic acid agarose. They have low affinities for the usual penicillins and cephalosporins.