Platelet-rich plasma (PRP), a platelet concentrate made of autogenous blood, has been used in recent years to improve bone defect healing particularly in maxillofacial reconstructions. The aim of the present study was to assess the effect of PRP on new bone formation in a critical diaphyseal long bone defect. A critical size defect (2.5 cm) in the tibial diaphysis of 16 sheep was supplied either with autogenous PRP in a collagen carrier or with collagen alone (controls). Platelets were enriched about 3.5 fold compared to normal blood in the PRP. After 12 weeks, the explanted bone specimens were quantitatively assessed by X-ray, computed tomography (CT), biomechanical testing and histological evaluation. Bone volume, mineral density, mechanical rigidity and histology of the newly formed bone in the defect did not differ significantly between the PRP treated and the control group, and no effect of PRP upon bone formation was observed. It was suggested that PRP does not enhance new bone formation in a critical size defect with a low regenerative potential. Such bone defects might require more potent stimuli, e.g. combinations of functional biomaterials or autografts, precursor cells or osteoinductive growth factors.