1,25-Dihydroxyvitamin D-3 [1,25(OH)(2)D-3] initiates the hydrolysis of sphingomyelin in ROS 17/2.8 osteosarcoma cells with the resultant generation of cell-associated ceramide. Increases in ceramide levels were detectable at; 15 min and maximal one hour after exposure of cells to 1,25(OH)(2)D-3. Neither 1,25(OH)(2)D-3 nor exogenous ceramide elicited a change in cytosolic free Ca2+ ([Ca2+](i)). Transient elevations in [Ca2+](i) were observed when cells were exposed to exogenous sphingosine, but there was no detectable conversion of ceramide to sphingosine in 1,25(OH)(2)D-3-treated cells. Ceramide also did not stimulate Ca2+ uptake across ROS 17/2.8 cell plasma membranes. Collectively, these results suggest that 1,25(OH)(2)D-3 activates sphingomyelin turnover in ROS 17/2.8 osteosarcoma cells but that the sphingolipid metabolite ceramide is not responsible for 1,25(OH)(2)D-3-induced activation of plasma membrane Ca2+ channels.