Transcription factor p53 regulates the cell cycle and apoptosis and may impair angiogenesis by the deregulation of pro-angiogenic factors and the activation of anti-angiogenic factors. Our aim has been to elucidate further the role of p53 in physiological angiogenesis. By treating hamsters with the wildtype p53 inhibitor pifithrin-alpha (PFT) versus equivalent volumes of the vehicle dimethylsulfoxide, we showed a reduced p53 tissue protein level, a reduction of poly(ADP-ribose) polymerase and cleaved caspase-3 products, and a slightly increased proliferation of cell nuclear antigen and cyclin D1 by Western blot protein analysis of ovarian tissue. PFT further increased platelet-derived growth factor and did not influence vascular endothelial growth factor in female reproductive tissue. Despite these differences in tissue levels of proteins potentially involved in angiogenesis, in vivo fluorescence-microscopic analysis of freely transplanted ovarian follicles revealed comparable kinetics and an extent of revascularization with almost identical densities of network microvessels in both groups. However, follicles of PFT-treated animals exhibited enlarged diameters and higher volumetric blood flow within the newly formed microvessels. Less-dense basement membranes with unclear laminar structure and only a loose contact of pericytes to endothelial cells were also occasionally found, providing evidence of delayed maturation and impaired diameter control of microvessels. Thus, inhibition of wildtype p53 during physiological angiogenesis does not affect the extent of new vessel formation but may delay the maturation of newly formed microvessels.