Bacterial infection in the vicinity of guided tissue regeneration barrier membranes was shown to have a negative effect on the clinical outcomes of this increasingly used technique. Several oral and specifically periodontal bacteria were shown to adhere to such membranes in vivo and in vitro with a higher affinity to membranes constructed from collagen. The present study examined the role of periodontal bacteria and their enzymes in the degradation of commercially used collagen membranes. Degradation of two collagen membranes [Biomend (Calcitek, Colla-Tec Inc., Plainsboro, NJ) and Bio-Gide (Geistlich Biomaterials, Wolhousen, Switzerland)] labeled by fluorescein isothiocyanate was examined by measuring soluble fluorescence. Porphyromonas gingivalis, Treponema denticola and Actinobacillus actinomycetemcomitans and their enzymes were evaluated. Collagenase from Clostridium hystolyticum was used as a positive control. While whole cells of P. gingivalis were able to degrade both types of membranes, T. denticola could degrade Bio-Gide membranes only and A. actinomycetemcomitans whole cells could degrade none of the membranes. Fractionation of P. gingivalis cells revealed that cell membrane associated proteases were responsible for the degradation of the two collagen membranes. In T. denticola, the purified major phenylalanine protease was found to be responsible for the degradation of Bio-Gide membranes. These results suggest that proteolytic bacterial enzymes may take part in the degradation of collagen barrier membranes used for guided tissue regeneration.