Spirochetes are usually the predominant organisms observed microscopically in subgingival plaques removed from tooth sites associated with periodontitis, but these organisms are rarely isolated by cultural means, presumably because the media do not support their growth and/or because these fragile organisms are disrupted by the various procedures used to disperse plaque samples. In the present investigation, three dispersal procedures, sonification, mechanical mixing, and homogenization, were compared for their ability to permit the isolation of Treponema denticola, Treponema vincentii, Treponema socranskii, and Treponema pectinovorum from plaque samples on media that support the growth of these species. Plaque samples in which the spirochetes averaged 50% of the microscopic count were chosen. The highest viable recoveries of spirochetes were observed when the plaques were dispersed with a Tekmar homogenizer, and the lowest occurred with sonification. The highest recoveries averaged only about 1% of the total cultivable counts, indicating either that the sought-after species were minor members of the flora or that the dispersal procedures were still too harsh. A total of 91% of the isolates were T. denticola, 5% were T. socranskii, and 4% were a heretofore-undescribed species.