The acute effects of insulin and adenosine on rates of lipolysis and lipogenesis in pig adipocytes were investigated to determine what limits the expression of the insulin response in vitro. Adenosine and insulin independently inhibited isoproterenol-stimulated lipolysis. Adenosine, acting through the pertussis toxin-sensitive G-protein Gi, was more effective than insulin and could completely inhibit lipolysis. Fatty acid synthesis from glucose was increased by both adenosine and insulin. Neutralization of endogenous adenosine with adenosine deaminase decreased basal rates of lipogenesis and increased the insulin response from 30 to 60% above basal. Neutralization of Gi with pertussis toxin further decreased the basal rate and increased the insulin response to 160% above basal. These data indicate that Gi, and the ligands that signal through Gi, stimulate glucose incorporation into fatty acids and can attenuate the insulin response. It seems likely that an exaggerated rate of glucose metabolism in the absence of insulin contributes to the inconsistent insulin responses exhibited in pig adipose tissue in vitro. These data also demonstrate that insulin and adenosine have major roles in regulating pig adipose tissue metabolism.