Experimental heterotopic bone formation was produced by subcutaneous implants of demineralized allogeneic bone matrix (DABM) in vitamin D-deficient (-D) animals that were either not treated or given vitamin D3 (+D) or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to determine the role of vitamin D and its most active metabolite in osteoinduction and implant remodeling. Histologically, implants in both +D and -D groups caused a similar acute inflammatory response, formation of a fibrous capsule, and chondrogenesis by 1 to 2 weeks after implantation. However, by 3 weeks after implantation implants in the -D animals had formed less bone matrix, had developed a defect in matrix mineralization, had reduced bone forming and bone resorbing surfaces, and had altered bone architecture resulting from defective bone remodeling. The altered histology in -D animals was not corrected by 10 weeks after implantation. Treatment of vitamin D-deficient rats with 1,25(OH)2D3, 65 pmol/day for 3 weeks, corrected both the defect in mineralization and the abnormal histology. The results indicate that (1) vitamin D deficiency does not alter either the timing or the sequence of histologic events associated with osteoinduction but dramatically reduces the magnitude of the response, (2) vitamin D deficiency not only impairs mineralization but also reduces bone formation and resorption, and (3) 1,25(OH)2D3 mimics all of the actions of vitamin D with regard to correcting the abnormal osteoinductive response and bone histomorphometry.