A phosphono analog of choline phosphoglyceride was used to examine the relative contributions of phospholipase C and D in the generation of diglycerides in fMLP- and A23187-stimulated human neutrophils. The phosphono analog, 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphonocholine, contains a carbon-phosphorus bond adjacent to the base moiety and is resistant to phospholipase D hydrolysis, while remaining susceptible to phospholipase C hydrolysis. fMLP stimulated the production of [3H]phosphatidic acid and subsequently [3H]diglyceride from cells containing 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine, but not from cells prelabeled with the phosphono analog. Treatment with A23187 also resulted in the formation of these products from cells containing 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine. Additionally, A23187 stimulated the conversion of the phosphono analog to phosphodiester-containing choline phosphoglyceride which then resulted in the generation of [3H]phosphatidic acid and subsequently [3H]diglyceride. This study demonstrates the use of a phosphono analog in assessing phospholipase C and D activity in cells and provides evidence that in fMLP- and A23187-stimulated human neutrophils, diglyceride is generated indirectly from choline phosphoglycerides by the combined activities of phospholipase D and phosphatidate phosphohydrolase.