Calcification of the vessel wall is a regulated process with many similarities to osteogenesis. Progenitor cells may play a role in this process. Previously, we identified a novel gene, Vascular Calcification Associated Factor (VCAF), which was shown to be important in pericyte osteogenic differentiation. The aim of this study was to determine the localization and expression pattern of VCAF in human cells and tissues. Immunohistochemical analysis of seven atherosclerotic arteries confirmed VCAF protein expression within calcified lesions. In addition, individual VCAF-positive cells were detected within the intima and adventitia in areas where sporadic 3G5-positive pericytes were localized. Furthermore, VCAF-positive cells were identified in newly formed microvessels in association with CD34-positive/CD146-positive/c-kit-positive cells as well as in intact CD31-positive endothelium in internal mammary arteries. Western blot analysis confirmed the presence of VCAF (18 kD) in protein lysates extracted from human smooth muscle cells, endothelial cells, macrophages, and osteoblasts. In fracture callus samples from three patients, VCAF was detected in osteoblasts and microvessels. This study demonstrates the presence of VCAF in neovessels and raises the possibility that VCAF could be a new marker for vascular progenitor cells involved in a number of differentiation pathways. These data may have implications for the prevention or treatment of vascular disease. Copyright © 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.