Pyocin inhibition of Neisseria gonorrhoeae and its feasibility as a gonococcal typing scheme were examined. Mitomycin C-induced pyocin lysates of Pseudomonas aeruginosa were able to selectively inhibit the growth of gonococcal strains. The particles associated with the inhibitory activity were non-dialyzable, heat labile, Pronase sensitive, trypsin resistant, and of large molecular weight by membrane and gel filtration techniques. The inhibitory activity was shown to be specific by absorption with sensitive and insensitive strains of N. gonorrhoeae and P. aeruginosa. Partial purification of pyocin lysates by ammonium sulfate precipitation followed by ultracentrifugation revealed phagelike particles consistent with high-molecular-weight R-type pyocines. These particles were associated with increased inhibitory activity and could be seen associated with the gonococcal cell surface. One hundred and six gonococcal strains could be differentiated on the basis of their sensitivity of 23 pyocin extracts. Thirty different patterns of pyocin inhibition were seen. Isolates from different body sites from the same patient could generally be identified as being similar strains. Strains isolated from known consorts had the same patterns. In general, agreement between pyocin typing and available epidemiological information was good.