The prominent purpose of the study was the evaluation of the in vitro mitogenic effect of three different homologous platelet-rich plasma (PRP) preparations (PRPa, PRPb, PRPc) on three different lines of periodontal ligament (PDL) cells (PDL(1,2,3)), cultured alone or in combination with a demineralized freeze-dried allograft (DFBA). PDL cell cultures were derived from the mid root of three maxillary caries-free premolars extracted for orthodontic reasons. Cells were grown and reached confluence. To evaluate the mitogenic effect of all exogenous factors (PRPa, PRPb, PRPc and DFBA) on PDL cells, specific number of cells (10.000/well) was cultured in the presence or absence of the above factors. Each PRP preparation (5% v/v) was added in all cell lines, in the absence or presence of 10 mg/ml of DFBA. The cells were also treated with 25 ng/ml bFGF (positive control). The mitogenic effect was evaluated 24 h after incubation, using the Trypan blue exclusion assay. The results revealed that all PRP preparations act as potent mitogens as they significantly induced cell proliferation on PDL(1,2,3) lines. All PRP preparations when added alone in the PDL cell cultures, exhibited a significant advantage over the positive control (bFGF). The addition of DFBA to PRP did not influence significantly cell proliferation in all cell lines, comparatively to PRP alone, at the time -period studied. The findings of this study demonstrate the beneficial role of PRP alone or combined with the bone graft on periodontal ligament cells in vitro, suggesting that it may be considered as a potential biological approach in periodontal regeneration.