Nutritional experiments with growing rats were conducted to study the effect of dietary t18:1 and t,t18:2 in mixtures with safflower oil (77.1% linoleate) on oxidative phosphorylation of isolated liver mitochondria and on energetic efficiency as measured in energy balance experiments. Partially hydrogenated soybean oil (PHSO) with 45% t18:1 and an ethyl ester concentrate of trans fatty acids (TRANS) containing 52% t,t18:2 were used. Six groups of male rats were fed diets with 10% (wt/wt) fat supplements: 1, safflower oil (SAF); 2, 8% PHSO + 2% SAF; 3, 0.5% TRANS + 9.5% SAF; 4, 1% TRANS + 9% SAF; 5, 2% TRANS + 8% SAF; and 6, 5% TRANS + 5% SAF. These fat supplements contained 77.1, 17.1, 73.3, 69.4, 61.7 and 38% linoleate for groups 1-6, respectively. The basal metabolic rate was not affected by dietary treatments, neither were the metabolizable energy values. As compared to the SAF group, rats receiving the 8% PHSO + 2% SAF showed no different effect on energy utilization or mitochondrial respiratory function. TRANS was found to reduce the efficiency of metabolizable energy utilization at the 5% level (group 6), while mitochondrial ATP synthesis was significantly depressed in both the 2% and the 5% TRANS groups. A parallelism was observed between t,t18:2 incorporation in liver mitochondria, reduced mitochondrial oxidative function and depressed energetic efficiency. The experiments indicated that t,t18:2 exerted a significantly negative effect on energy utilization at 1.0 and 2.5% levels in the diet even when fed with relatively high concentrations of linoleate.