In isolated rat mammary secretory cells, insulin stimulated fatty acid synthesis from pyruvate three times, stimulated glucose conversion to fatty acids 1.2 to 1.5 times, and decreased lactate conversion to fatty acids 20 to 30%. Incubation of glucose and pyruvate together depressed fatty acid synthesis from glucose not attributable to isotope dilution. Glucose stimulated conversion of pyruvate-2-14 carbon to fatty acids without significantly affecting pyruvate-1-14 carbon conversion to 14-carbon dioxide. At differing concentrations, the electron acceptors phenazine methosulfate and N,N,N',N'-tetramethyl-p-phenylene-diamine alleviated the depression by insulin of lactate conversion to fatty acids. The data support concepts that: (1) insulin acts at important sites other than or in addition to glucose transport in regulating mammary secretory cell metabolism and, particularly, fatty acid synthesis; (2) insulin actions upon fatty acid synthesis can vary dependent upon cellular redox state (insulin increases fatty acid synthesis in cells with a low redox state and decreases fatty acid synthesis in cells in a very reduced state); and (3) pyruvate depresses glucose carbon flux through the Embden-Meyerhof pathway.