1 alpha,25-Dihydroxyvitamin D3 rapidly increases cytosolic calcium and alters membrane phospholipid metabolism in hepatocytes. To define the causal relationship between these events, we examined the effects of 1 alpha,25-dihydroxyvitamin D3 on 32P-labeled lysophosphatidylinositol levels and cytosolic calcium as affected by pertussis toxin and 1 beta,25-dihydroxyvitamin D3, the biologically inactive analog. 32P-labeled lysophosphatidylinositol was determined by two-dimensional thin-layer chromatography. Cytosolic calcium was measured in cells loaded with quin-2AM. Within 5 min, 1 alpha,25-dihydroxyvitamin D3 increased hepatocyte cytosolic calcium by 31% (p less than 0.05) and 32P-labeled lysophosphatidylinositol by 38% (p less than 0.05). Pertussis toxin inhibited the hormone-induced rise in cytosolic calcium but not the increase in 32P-labeled lysophosphatidylinositol. Exposure to exogenous lysophosphatidylinositol for 5 min increased cytosolic calcium by 40% (p less than 0.05), an effect that was also inhibited by pertussis toxin. 1 beta,25-Dihydroxyvitamin D3 had no effect on either hepatocyte cytosolic calcium or 32P-labeled lysophosphatidylinositol but prevented the 1 alpha,25-dihydroxyvitamin D3-induced increments. The results suggest that a G protein sensitive to pertussis toxin is required for the transduction of the lysophosphatidylinositol signal but not the generation of the signal. The ability of 1 beta,25-dihydroxyvitamin D3 to inhibit the 1 alpha,25-dihydroxyvitamin D3-induced changes in phospholipids suggests that the epimer may compete with 1 alpha,25-dihydroxyvitamin D3 for an initiating receptor.