Mammary gland utilizes a combination of substrates in vivo. To determine if and how the substrates presented to the gland alter milk fat synthesis, slices of mammary gland of lactating rats were incubated with combinations of glucose, L-lactate, and pyruvate. Uptakes and conversions of uniformly and specifically labeled substrates to CO2 and fatty acid were measured. The absolute rate of fatty acid synthesis was measured by incoporation of tritium from tritiated water into fatty acid. The extent, but not the type, of fatty acids synthesized was affected by the substrates utilized. Glucose stimulated uptake and conversion to fatty acid of l-lactate and, to a smaller extent, of pyruvate. Analysis of CO2 and fatty acid yields revealed that (a) the major stimulatory effect of glucose was on the conversion of acetyl coenyzme A from other substrates to fatty acid; (b) the rate of fatty acid synthesis paralleled the activity of the hexose monophosphate pathway up to a point. A likely source of the NADPH required beyond this point is provided by oxidation of malate to pyruvate. Reduction of NADH of oxaloacetate, produced during citrate cleavage, would yield malate. Hence, maximal synthesis would require production of NADPH and NADH, a condition met when glucose and L-lactate were substrates. Decreased fatty acids synthesis in pyruvate's presence supports this suggested requirement for cytosolic NADH in rat mammary gland.