The fastidious gram negative asaccharolytic rods Wolinella recta, Campylobacter concisus, Bacteroides gracilis, and Eikeneila corrodens have been isolated from lesions of advanced periodontal disease. In this investigation, three methods were compared for their ability to identify fresh isolates of these species: 1) biochemical tests and sensitivities to dyes and other inhibitors, 2) serological reactions, and 3) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). SDS‐PAGE of total cell proteins appeared to be the most sensitive and accurate method to identify isolates, although many strains were successfully identified using serology. Biochemical tests including sensitivities to inhibitors were the least reliable. W. recta, B. gracilis, and E. corrodens were found more frequently and in higher numbers in progressive (active) disease sites of many of the subjects. E. corrodens was also found to be significantly elevated in inactive sites of certain subjects. After successful treatment with Widman flap surgery and systemically administered tetracycline, the proportions of W. recta and E. corrodens were reduced, while those of C. concisus were elevated.