OBJECTIVE Regenerative periodontal therapy using platelet-rich plasma (PRP) and bone substitutes with guided tissue regeneration (GTR) have been proposed as a therapeutic method to enhance the outcome of regenerative surgery. This includes light microscopic evaluation of retrieved ePTFE membranes to assess formation of new connective tissue attachment, and following the regeneration process. The objectives of this study were to evaluate the histological findings of retrieved ePTFE membranes using PRP and bone substitutes, the effect of PRP on graft materials, and the correlation of the findings with the clinical outcomes. METHODS Seventy-two (72) patients with chronic periodontitis, each of whom had one deep intrabony defect, were randomly included in two studies and treated using the same type of membrane and different bone substitutes. In the first study, 17 cases were treated with a natural bone mineral and a non-resorbable membrane (NBM + GTR), and 17 cases were treated with PRP + NBM + GTR. In the second study, 19 patients were treated with β-tricalcium phosphate and a non-resorbable membrane (β-TCP + GTR), and 19 patients were treated with PRP + β-TCP + GTR. In both studies, tissue integration of the retrieved ePTFE membranes and attached remnants were evaluated histologically. RESULTS Histological scores showed that membranes combined with NBM are better integrated than membranes combined with β-TCP; the difference between the two decreased with the addition of PRP. The application of PRP had no significant effect on the quality of membrane integration combined with NBM, whilst significantly improving the integration quality when combined with β-TCP. No correlations were detected between the histological scores and the clinical attachment level (CAL) gain in any of the groups. CONCLUSIONS The present results indicate that: a) application of β-TCP and PRP may enhance membrane integration and periodontal healing, and b) histological examination of retrieved membranes may provide valuable additional information with regard to the clinical findings.