The influence of the growth medium on the ability of strains of Streptococcus mutans, Actinomyces viscosus and A. naeslundii to attach to saliva-treated hydroxyapatite (S-HA) surfaces was studied. Preliminary experiments indicated that cells of each species harvested in lag, log, and early stationary phases of growth adsorbed comparably to S-HA; thus, early stationary phase cells were used in all subsequent assays. Strains were grown in chemically defined medium (CDM), in CDM supplemented with gastric mucin or with filter-sterilized or (60)Co-irradiated saliva from human donors of blood types A, B, or O, and in Trypticase soy broth (BBL Microbiology Systems) and Todd-Hewitt broth. Adherence of S. mutans H12 to S-HA tended to vary when the streptococci were grown in saliva-supplemented CDM, but the number of cells which attached was generally within twofold of that of CDM-grown cells. Attachment of A. viscosus S2 and LY7 and of A. naeslundii S4 and L13 was generally similar when grown in CDM or in CDM supplemented with saliva, but it tended to increase for organisms grown in CDM supplemented with gastric mucin. None of the strains studied appeared to destroy the blood group reactivity of the added salivary components, and they attached equally well to HA treated with homologous or heterogous saliva from that present in the medium in which they were grown. The A. viscosus strains adsorbed in 25 to 40% higher numbers to HA treated with blood type B saliva than with type A saliva, irrespective of the medium used for growth. S. mutans H12 cells displayed alpha- and beta-glucosidase and alpha-galactosidase activity; the Actinomyces strains exhibited these activities plus beta-galactosidase when grown in all media. However, the levels of these glycoside hydrolases did not correlate with cell adsorption to S-HA. The apparent weak influence of the growth medium on attachment of S. mutans was studied further. Strains of S. mutans isolated from the saliva of five human donors were made resistant to streptomycin, grown in CDM, and then added to new saliva samples from the respective donors from which they were obtained. The in vitro-grown cells were found to attach to S-HA comparably to S. mutans cells present naturally in the saliva.