The principal proteins associated with Neisseria gonorrhoeae peptidoglycan (PG), as identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, are the following: two proteins at approximately 90 kilodaltons (kDa), single major species at both 60 and 44 kDa, a 34- to 36-kDa protein, and three proteins between 28 and 32 kDa. A protein analogous to Escherichia coli Braun lipoprotein was not detected with gonococcal cell wall preparations. The identity of the PG-associated proteins was confirmed immunologically with antibody generated against purified cell walls. Two types of protein species, dithiothreitol extractable (the majority) and alkylation dependent (primarily the 34- to 36-kDa protein), appeared to be associated with the N. gonorrhoeae cell wall fraction. It was found that a crucial step in the extraction of the proteins from the PG fraction was the inclusion of an acetone-water wash of the purified PG pellet. Studies with cell wall preparations obtained from N. gonorrhoeae intrinsically labeled with 32P revealed that the acetone wash was removing phospholipid from the cell wall fraction and thus facilitating protein extraction. Autoradiographic analysis with PG material derived from 125I-surface-labeled cells indicated that the 44-kDa protein is exposed on the surface of the organism even when associated with the PG layer. Radioimmunoprecipitation with anti-PG antibody confirmed these findings. Lectin analysis (wheat germ agglutinin conjugated to horseradish peroxidase) suggested that the 34- to 36-kDa protein is covalently attached to the PG layer.