Angiogenesis is a pivotal event in endochondral ossification. Vessels grow into the hypertrophic cartilage and erode it to produce a scaffold on which osteoblasts settle to produce woven bone. A new culture system was used to determine whether growth-plate chondrocytes produce an angiogenic molecule. Chondrocytes from primary growth plates of bovine fetuses were separated into maturationally distinct subpopulations. When cultured these cells produce an extensive extracellular matrix and the prehypertrophic cells mature to express the hypertrophic phenotype defined by the synthesis of type X collagen and matrix calcification. The culture medium collected from the hypertrophic cells contains a chemoattractant, nonmitogenic molecule for bovine endothelial cells which can induce angiogenesis in vivo in the rabbit cornea model. This molecule has a Mr of approximately 120 x 10(3). The production of this molecule by hypertrophic cells is enhanced by both 1,25-(OH)2 vitamin D3 and 24,25-(OH)2 vitamin D3 at 10(-8)-10(-12) M, but only in pre- and early hypertrophic cells. In contrast, these metabolites have either no effect or an inhibitory effect on the more mature hypertrophic cells. These results describe for the first time the production of an angiogenic molecule by hypertrophic chondrocytes. They demonstrate an important role for vitamin-D3 metabolites in regulating hypertrophy and angiogenesis during normal skeletal growth and differentiation. Thus, a defective regulation of these processes, due to the lack of vitamin-D metabolites, may explain the observed enlargement of the hypertrophic zone and impairment of skeletal growth in rickets which is induced clinically and experimentally by a deficiency of vitamin D.