In this study, the interactions among breed of cattle, adipose tissue site and specific incubation conditions were investigated. Subcutaneous and i.m. adipose tissues were obtained from 10 Angus and 9 Santa Gertrudis steers immediately postmortem. Adipose tissue explants were incubated acutely for 2 h immediately at slaughter or after being cultured 48 h with or without 1 mU/ml insulin and 30 mg/ml bovine serum albumin; the incorporation of 14C-labeled acetate and glucose (5 mM, plus 5 mM unlabeled lactate) into lipid fractions was measured. AT the same chronological age, Angus steers had a more youthful lean maturity score, higher USDA marbling score and higher USDA quality grade (P less than .05) than did carcasses from Santa Gertrudis steers. The lower marbling score of the Santa Gertrudis steers was paralleled by smaller i.m. adipocytes (P less than .05) relative to Angus steers. Pentose cycle reductase and NADP-malate dehydrogenase activities were greater in Angus i.m. adipose tissue than in Santa Gertrudis i.m. adipose tissue, which would provide more reducing equivalents (NADPH) and glycerol for fatty acid biosynthesis and triacylglycerol esterification. Correspondingly, Angus i.m. adipose tissue exhibited a greater rate of lipogenesis from acetate and glucose (P less than .05) than did Santa Gertrudis i.m. adipose tissue in acute incubations. The presence of insulin resulted in higher rates of lipogenesis from acetate in Angus s.c. adipose tissue than in Santa Gertrudis s.c. adipose tissue after 48 h of explant culture. These data indicate that i.m. and s.c. adipose tissues exhibit aspects of lipid metabolism unique to each tissue and suggest that breed-related differences in adipose tissues may explain the divergent responses to insulin observed in different laboratories.