The purpose of the present investigation was to compare the composition of the predominant cultivable microbiota associated with gingival crevicular epithelial cells with that of the unattached microbiota recovered from the same site. Samples were taken from 2 diseased sites from 8 periodontal patients, by scraping the epithelial lining of the pocket with a curette. The epithelial cells were separated from the unattached subgingival bacteria by centrifugation in a reduced 50% Percoll density gradient. Epithelial cells formed a band at the top of the gradient and were removed separately from the unattached bacteria located at the base. Each layer was dispersed, diluted and plated on Trypticase soy agar with 5% sheep blood and 50 isolates were characterized from each sample. The microorganisms associated with the epithelial layer harbored 5- to 20-fold higher mean percentages of Bacteroides gingivalis, Bacteroides intermedius and Peptostreptococcus micros. The layer of unattached organisms exhibited 4- to 10-fold higher mean percentages of Streptococcus uberis, Capnocytophaga ochracea, Eikenella corrodens and Veillonella parvula.