Treatment of human HL-60 promyelocytic leukemia cells with phorbol-12-myristate-13-acetate (PMA), a tumor promoter and inducer of differentiation, stimulated the incorporation of label from L-[methyl-3H]methionine into the cellular phospholipids. Such a stimulation of phospholipid methylation was not observed in an HL-60 cell variant that is resistant to phorbol ester-induced differentiation. Enhanced methylation of phospholipids was detected 6 h after treatment and reached a maximum level of about twice the control level at 24-48 h. The degree of phospholipid methylation was dependent on the phorbol ester dose. The stimulation in phospholipid methylation by PMA was confirmed by measuring the activity of phosphatidylethanolamine methyltransferase in cellular lysates. After 24 or 48 h of exposure, the enzyme activity was elevated in the HL-60 cell lysates but not in the resistant cells. Phospholipid methylation was also stimulated after treatment of the HL-60 cells with the phorbol diester phorbol-12,13-dibutyrate or teleocidin, which is not a phorbol ester compound. These two chemicals and PMA are tumor promoters and inducers of cell differentiation in the HL-60 cells. Phorbol-12,13-diacetate and 4-O-methyl PMA, which are not tumor promoters or inducers of cell differentiation in the HL-60 cells, did not stimulate phospholipid methylation. The possible role of enhanced phospholipid methylation in cell differentiation of the HL-60 by these chemicals is discussed.