1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] receptors have been previously described in Sertoli cells. This study was performed to assess biological activity of the receptor in the mouse Sertoli cell line TM4. A 2-h preincubation with 0.01-25 nM 1,25(OH)2D3 resulted in a dose-dependent rapid uptake of 45Ca2+ within 5 min of addition of the isotope to the cells (27 +/- 8%, n = 4 experiments; P less than 0.05). This response was specific for 1,25(OH)2D3, in that it was not induced by 25-hydroxyvitamin D3, estradiol, cortisol, R 5020 (promegestone), or testosterone. However, a combination of testosterone and 1,25(OH)2D3 inhibited uptake by 23 +/- 8% (n = 3 experiments, P less than 0.01). That the mechanism responsible for 1,25(OH)2D3-stimulated uptake may involve 1,25(OH)2D3 receptor interaction is supported by the observation that cycloheximide inhibited the response. Conversely, there was no detectable change in uptake by 1,25(OH)2D3-treated cells after 24-h incubation with 0.1-5 nM 1,25(OH)2D3. Increased levels of DNA and protein content also resulted from a 2-h incubation with the steroid and were sustained up to 24 h without a concomitant increase in cell number or a detectable change in cell morphology. The presence of specific 1,25(OH)2D3 receptor-like binding sites was demonstrated by sucrose gradient analysis and hydroxylapatite assay. These data demonstrate that 1,25(OH)2D3 may play an important role in testicular function through regulation of receptor-mediated events.