The hormonal form of vitamin D, 1,25(OH)2 vitamin D3 [1,25(OH)2D3], regulates colonic calcium absorption and colonocyte proliferation and differentiation. In this study, we have examined the effect of 1,25(OH)2D3 on membrane phosphoinositide turnover, protein kinase C activation, and regulation of intracellular calcium concentration [( Ca+2]i) in isolated rat colonic epithelium. In a concentration-dependent manner, 1,25(OH)2D3 stimulated breakdown of membrane phosphoinositides within 15 s, generating diacylglycerol and inositol 1,4,5-triphosphate (IP3). 1,25(OH)2D3 rapidly activated colonic protein kinase C, with maximal translocation of activity from the cytosol to the membrane occurring within 1 min of exposure to the secosteroid. Studies performed in isolated colonocytes with the fluorescent dye fura-2 demonstrated that 10(-8) M 1,25(OH)2D3 caused a rapid rise in [Ca+2]i which then transiently decreased before rising to a new plateau value. When these experiments were performed in a calcium-free buffer, an increase in [Ca+2]i was observed, but both the transient and secondary rise were diminished in magnitude, suggesting that 1,25(OH)2D3 may stimulate both release of intracellular calcium stores and calcium influx. 1,25(OH)2D3 stimulated [3H]thymidine uptake in rat colonocytes, 4 h after an in vivo injection. These studies indicate that 1,25(OH)2D3 exerts a rapid influence on membrane phosphoinositide metabolism which may mediate certain of the secosteroid's effects on colonocyte calcium transport and proliferation.