Mice deficient in the expression of vitamin D binding protein (DBP) are normocalcemic despite undetectable levels of circulating 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃). We used this in vivo mouse model together with cells in culture to explore the impact of DBP on the biological activity of 1,25(OH)₂D₃. Modest changes in the basal expression of genes involved in 1,25(OH)₂D₃ metabolism and calcium homeostasis were observed in vivo; however, these changes seemed unlikely to explain the normal calcium balance seen in DBP-null mice. Further investigation revealed that despite the reduced blood levels of 1,25(OH)₂D₃ in these mice, tissue concentrations were equivalent to those measured in wildtype counterparts. Thus, the presence of DBP has limited impact on the extracellular pool of 1,25(OH)₂D₃ that is biologically active and which accumulates within target tissues. In cell culture, in contrast, the biological activity of 1,25(OH)₂D₃ is significantly impacted by DBP. Here, although DBP deficiency had no effect on the activation profile itself, the absence of DBP strongly reduced the concentration of exogenous 1,25(OH)₂D₃ necessary for transactivation. Surprisingly, analogous studies in wildtype and DBP-null mice, wherein we explored the activity of exogenous 1,25(OH)₂D₃, produced strikingly different results as compared to those in vitro. Here, the carrier protein had virtually no impact on the distribution, uptake, activation profile or biologic potency of the hormone. Collectively, these experiments suggest that while DBP is important to total circulating 1,25(OH)₂D₃ and sequesters extracellular levels of this hormone both in vivo and in vitro, the binding protein does not influence the hormone's biologically active pool.