The cardiac subtype of the L-type voltage sensitive calcium channel (VSCC), Ca_v1.2 (1_C), is the primary voltage sensitive channel responsible for Ca²⁺ influx into actively proliferating osteoblasts. This channel also serves as the major transducer of Ca²⁺ signals occurring in growth phase osteoblasts in response to hormone treatment. In this study, we demonstrate that 24 hr treatment of MC3T3-E1 pre-osteoblasts with 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), a coupling factor for bone resorption, coordinately down-regulates Ca_v1.2 (1_C) and uniquely up-regulates a T-type channel, Ca_v3.2 (1_H). No other voltage sensitive channel alpha subunit, of ten that were surveyed, was up-regulated by 1,25(OH)₂D₃. The shift from predominantly L-type to T-type channel expression is demonstrated to occur at both mRNA and protein levels detected by quantitative PCR and immunohistochemistry using antibodies specific for each channel type. Functional and pharmacological studies using specific inhibitors revealed that treatment with 1,25(OH)₂D₃ also altered the Ca²⁺ permeability properties of the osteoblast membrane from a state of primarily L-current sensitivity to one of T-current sensitivity. We conclude that the L-type channel is likely to support proliferation of osteoblast cells whereas T-type channels are more likely to be involved in supporting differentiated functions after 1,25(OH)₂D₃ mediated reversal of remodeling has occurred. This latter observation is consistent with the unique expression of the T-type VSCC, Ca_v3.2 (1_H), in terminally differentiated osteocytes that we recently reported.