Abstract Activating the protein-tyrosine kinase of v-Fps results in a rapid increase in diglyceride (DG) in rat fibroblasts. The v-Fps-induced increases in DG were detected only when phospholipids were prelabeled with [ 3H]-myristate, which is incorporated primarily into phosphatidylcholine (PC). Inhibition of phosphatidic acid (PA) phosphatase (PAP), which converts PA to DG, blocked v-Fps-induced DG production. PA is a primary metabolite of type D phospholipases (PLD). Consistent with these observations, PLD activity was activated in response to the kinase activity of v-Fps. The increased PLD activity was detected only when the cells were prelabeled with the PC-specific [ 3H]-myristate. These data support the hypothesis that v-fps-induced DG is derived from PC via the PLD/PAP pathway.