Human hairy cell leukemia (HCL) cells in culture showed a marked increase in both [1-14C]acetate and [14C]choline incorporation into phosphatidylcholine (PC) when treated with a 10 nM concentration of 12-O-tetradecanoylphorbol 13-acetate (TPA) for 3 h. Dramatic morphological changes occurred and synthesis of most phospholipids was stimulated. However, the most dramatic increase was seen in the [14C]acetate labeling of both long- and short-chain fatty acid-containing sphingomyelins (from 200-425% of control levels), sphingomyelin being especially enriched in HCL cells. Negligible incorporation of [14C]choline into sphingomyelin was observed and phospholipase inhibitor (U10029A) studies indicated that PC was the major source of sphingomyelin choline. These changes were most clearly seen by autoradiography of two-dimensional thin-layer chromatography plates. Chronic myelogenous leukemia (CML) blasts, which did not respond morphologically to TPA, showed no increased phospholipid synthesis under the same conditions and increases in sphingomyelin synthesis were modest. Other non-TPA-responding leukemic cells were similarly refractive. However, one out of four acute monomyelocytic leukemic (AMMoL) cells studied responded morphologically in a manner identical to HCL cells and exhibited the same dramatic increase in sphingomyelin synthesis. Data are presented which suggest that TPA may also stimulate PC phospholipase C activity in addition to activating the calcium-dependent protein kinase by mimicking diacylglycerol.