Protein I (PI), the major outer membrane protein of Neisseria gonorrhoeae, is a porin and occurs in two major immunochemical classes, A and B. By using shuttle mutagenesis to insert a selectable marker close to the PI structural gene, evidence was obtained from transformation experiments to demonstrate that the PI structural gene is equivalent to the defined locus nmp and that the genes for PI class A and PI class B are alleles of the same locus. The PI class B gene of strain MS11 was cloned and sequenced, and comparison of this sequence with the gene sequence of PI class A of FA19 revealed a number of regions of significant divergence. By selection for the closely linked marker in transformations between the two strains, a series of strains with a hybrid PI was obtained. Analysis of these strains with monoclonal antibodies and oligonucleotides specific to PI class A or PI class B elucidated the nature and location of some of the surface-exposed epitopes, a thorough characterization of which is a prerequisite for understanding the role of PI in gonococcal pathogenesis and its possible use as a component of a vaccine.