Osteopetrosis is a heterogeneous group of metabolic bone disorders characterized by reduced bone resorption. In the toothless (tl) osteopetrotic rat mutation there are few osteoclasts and mutants are not cured by bone marrow transplants. This suggests that the defect(s) in tl rats is within the skeletal microenvironment and not one of stem cell incompetence. Osteoblasts are known to play a role in bone resorption and abnormalities in these cells have been reported in tl rats. We explored the ability of osteoblasts from tl rats to activate resorption by normal osteoclasts when co-cultured in the presence of 1,25-dihydroxyvitamin D (1,25(OH)2D). Stimulation with 1,25(OH)2D produced a highly significant response in normal osteoblast co-cultures, but no response was observed in mutant cultures over a wide dose range. Ligand-binding studies demonstrated no abnormalities in vitamin D receptor (VDR) affinity, but mutant osteoblasts had reduced VDR numbers. Taken together with the demonstrated resistance of these mutants to the hypercalcemic effects of 1,25(OH)2D and parathyroid hormone in vivo, these data implicate osteoblasts in the pathogenesis of this mutation.