We investigated the in vivo effects of dietary conjugated linoleic acid (CLA) on subcutaneous adipose tissue from heavy pigs to clarify the involvement of possibly different causative effects in the established antiadipogenic effect of CLA. Pigs [n = 36; initial body weight, 106 kg live weight (LW)] were assigned to 1 of 2 LW-matched groups supplemented with either 0 or 0.75% of a CLA preparation containing 50% CLA isomers. The pigs were slaughtered at 155 kg LW and adipose tissue analyzed. CLA supplementation affected ash content, and decreased iodine values (P < 0.01) and adipocyte size (P < 0.05). The fat content of adipose tissue was lower (P < 0.05) in females than castrated males, and females had smaller (P < 0.01) adipocytes than castrated males. Neither CLA nor sex influenced adipocyte lipid droplet diameter or the extent of lipid peroxidation as determined by quantitation of Schiff's histochemical reaction. NADPH-diaphorase was not influenced by CLA treatment. Preadipocyte proliferation rates were lower in pigs fed CLA (P < 0.05), whereas the number of adipocyte apoptotic nuclei was greater (P < 0.05). Preadipocyte proliferation was also greater (P < 0.05) in females than castrated males. Neuronal and endothelial nitric oxide synthase activities did not differ between groups in adipose tissue vessels, but inducible nitric oxide synthase expression in adipocytes was lower in pigs fed CLA (P = 0.05). These findings suggest that the antiadipogenic effect of CLA in heavy pigs is not a direct effect but may occur by downregulation of a NO-mediated lipolytic pathway.