There is evidence that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] affects phospholipid metabolism of intestine, kidney, and bone. There are no such studies concerning the parathyroid gland, which is also a target tissue for 1,25-(OH)2D3. In this investigation we examined the effect of 1,25-(OH)2D3 on the incorporation of radiolabeled choline, inositol, serine, and ethanolamine into the phospholipids of cultured bovine parathyroid cells. Treatment with 10-8 M 1,25-(OH)2D3 for 48 h caused a significant decrease in [14C]choline incorporation, although there were no differences in the incorporation of radiolabeled inositol, serine, or ethanolamine. Time-course and dose-response evaluations of the 1,25-(OH)2D3 effect revealed that the decrease in [14C]choline incorporation was seen within 12 h of incubation and occurred with as little as 10-9 M, respectively. In contrast, neither 10-7 M 25-hydroxyvitamin D3 nor 10-7 M 24,25-(OH)2D3 caused significant changes in [14C]choline incorporation. When 5 × 10-6 M cycloheximide was added to the medium, the inhibitory effect of 1,25-(OH)2D3 on [14C]choline incorporation was completely abolished. A significant decrease in phosphatidylcholine content was observed after treatment with 10-8 M 1,25- (OH)2D3 for 96 h. 1,25-(OH)2D3 did not cause a dramatic change in the fatty acid composition of the phosphatidylcholine. The present studies demonstrate that in parathyroid cells 1,25- (OH)2D3 causes a decrease in [14C]choline incorporation, which could be due to a decrease in the synthesis of phosphatidylcholine or increased degradation. This effect is specific for 1,25- (OH)2D3 and requires new protein synthesis.