Concentrations of tolbutamide from 0.25 to 4.0 mg. per milliliter were found to inhibit epinephrine-stimulated lipolysis in isolated fat cells of the rat. At a concentration of 1 mg. per milliliter, tolbutamide also inhibited lipolysis stimulated by ACTH or dibutyryl cyclic AMP. At the same concentration it failed to alter basal lipolysis, basal or epinephrine-stimulated cyclic AMP levels. Basal and epinephrine-stimulated adenylate cyclase and glycogen phosphorylase as well as binding of cyclic AMP to protein kinase were unaltered. These data are discussed in relation to the antilipolytic mechanism of tolbutamide.