The in vivo effects of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] on cellular structure and response, matrix metachromasia and mineralization have been studied in the epiphysis and growth plate of humeri in normal neonatal mice. A relatively low dose of the metabolite, 50 ng/kg body weight, significantly increased the overall size of humeral growth plate, the zone of proliferating cells and that of hypertrophic chondrocytes. The response of the tissue to the metabolite changed with the increase in dose administered so that it was only the zone of proliferation that still showed increases in size in comparison to untreated controls. 1,25(OH)2D3 led to an increase in the metachromatic reaction of the cartilage matrix in the chondroblastic zone, and to marked increase in matrix mineralization in the hypertrophic zone. Qualitative changes were also noted in the structure of chondrobalsts and hypertrophic chondrocytes. 1,25(OH)2D3 affected the osteoblastic and osteocytic populations of cells along the metaphysis and diaphysis of the humerus. High doses of 1,25(OH)2D3 brought about distinct atrophic changes in the above cells. Chondrocytes in the epiphysis were found to synthesize type I collagen and fibronectin. These findings indicate that excessive doses of 1,25(OH)2D3 in an intact growing animal affect the normal differentiative pathway of prechondroblasts and thereby affect long bone development.