Oxidation of fatty acids was studied in bovine mammary tissue slices in order to evaluate their potential contribution to energy metabolism. Rates of fatty acid oxidation decreased with increasing chain length: acetate > octanoate > palmitate or oleate. Rates of oxidation of long chain, but not short chain, fatty acids increased over time, which could not be explained by carnitine palmitoyltransferase (CPT) activity. This phenomenon is not an artifact of the incubation system or caused by substrate solubility, as rates of palmitate oxidation were constant in rat kidney cortex slices. Preincubating mammary tissue with or without unlabeled palmitate showed that increasing rates of palmitate oxidation is not caused by use of endogenous fatty acids. Palmitate at 0.26 mM, equivalent to arterial fatty acid concentration, gave maximal rates of oxidation. The &oxidation enzymes may restrict fatty acid oxidation as oxidation of [ 1-14 C] palmitate exceeded that of [U-14C] paimitate. Acetate inhibited palmitate oxidation (75%) but not esterffication, suggesting that acetate inhibits palmitate oxidation by substrate competition at the mitochondrial level or via malonyl-CoA inhibition of CPT. Glucose inhibited palmitate oxidation (67%) and stimulated esterification. Low palmitoyl-CoA levels would favor glyceride synthesis over oxidation, since the apparent K m for palmitoyl-CoA of the glycerol-3phosphate acyltransferases is lower than that for CPT. Thus, glucose presumably diverts palmitate from oxidation to glycerolipids. Clofenapate, a glyceride synthesis inhibitor, decreased triacylglycerol formation, and marginally increased palmitate oxidation. We estimated that long chain fatty acids can potentially account for 6-10% of the oxidative metabolism of mammary tissue.