Insulin is known to be an important osteotropic hormone. To date, no study has specifically addressed the possibility that insulin receptor expression may be regulated by differentiation in bone. We report a heterogeneous distribution of insulin receptor (IR) within neonatal rat calvaria using a specific monoclonal antibody to the β‐subunit of the rat insulin receptor (CT‐1). Specific binding of CT‐1 to mature osteoblasts was demonstrated, with little binding over periosteal tissues or osteocytes. Using enzymatically derived subpopulations of calvarial cells, we showed a correlation between alkaline phosphatase activity and insulin‐stimulated 2‐deoxyglucose (2‐DOG) uptake and increased ¹²⁵I‐insulin binding. Since primary calvarial cultures contain many cell types, we compared ¹²⁵I‐insulin binding, insulin‐stimulated 2‐DOG uptake, and Northern blot analysis of IR mRNA in the clonal preosteoblast‐like cell line UMR 201‐10B and the mature osteoblast cell line UMR 106‐01. It is shown that UMR 106‐01 cells possess higher levels of IR mRNA, insulin binding, and insulin‐stimulated glucose uptake, and that insulin up‐regulated expression of mRNA of the glucose transporter GLUT1 by 3‐fold. In contrast, insulin binding was negligible in UMR 201‐10B cells, which expressed much lower levels of IR mRNA. UMR 201‐10B cells did not possess an insulin‐sensitive glucose uptake system, although they express GLUT1 mRNA. These data are consistent with the hypothesis that, as in muscle and fat, insulin receptor expression correlates with the stage of osteoblast differentiation in vivo and in vitro.