Cultured human promyelocytic leukemia cells were incubated with [1-14C]acetate to assess the predominant mode of fatty acid synthesis (de novo vs. chain elongation) in these cells and in chemically differentiated populations. 12-O-Tetradecanoylphorbol-13-acetate (TPA) and dimethyl sulfoxide (DMSO) will induce differentiation of this leukemic cell line to macrophage- and granulocyte-like cells, respectively. Human peripheral and rat peritoneal macrophages were employed as controls for the TPA-induced counterpart. Labeling of the carbonyl carbon (C-1) was determined by Schmidt degradation and used to distinguish between chain elongation of pre-existing cellular fatty acids and de novo synthesis. Cultured leukemia cells and the TPA-derived macrophage only incorporated from 16% to 23% of the total radioactivity into the C-1 position, indicating an operable de novo pathway. Cells differentiated by exposure to DMSO displayed a preference for the chain elongation mechanism (89% 14C in C-1 position) of fatty acid synthesis. Both rat peritoneal and human peripheral macrophages likewise incorporated greater than 80% of the radioactivity in the C-1 position of the fatty acyl chains. Thus, DMSO-treated leukemia cells resemble normal differentiated cells, whereas phorbol ester-induced cells, in contrast, retain biochemical features of the undifferentiated cancer cell.