One advanced localized periodontal lesion in each of 10 patients was treated using the guided tissue regeneration procedure. Six weeks after placement of expanded polytetrafluoroethylene (ePTFE) membranes, microbial samples were taken from each treated site and the membranes were removed. Six weeks later the cases were re-evaluated. They had all healed successfully with varying amounts of gain of clinical attachment. Gram-negative, anaerobic rods were found in all samples and made up 31% of all organisms cultivated. In 1 patient, Porphyromonas gingivalis was found in a proportion of 17.5%. Six of the other 9 patients harbored Prevotella intermedia (mean proportion 21.3%) and 6 Prevotella melaninogenica (6.8%). Fusobacterium and Capnocytophaga were also frequently found. The results demonstrate that ePTFE membranes are frequently colonized by periodontal microorganisms. The importance of bacterial colonization on clinical success is presently not known. Further studies are needed to determine the effect of the presence or absence of putative pathogens during guided tissue regeneration.