The interactions of polyamines with the lipolytic system were studied in isolated rat adipocytes. Spermine, spermidine and putrescine significantly inhibited adenosine deaminase-stimulated lipolysis. An antilipolytic effect of spermine was detectable at a concentration of 0.25 mM (P less than 0.05). At a concentration of 10 mM all three polyamines inhibited the stimulated lipolysis by 50-60% (P less than 0.001). In addition, spermine enhanced the antilipolytic sensitivity of insulin. Spermine (1 mM) decreased the half-maximal inhibitory concentration of insulin from 320 +/- 70 pM to 56 +/- 20 pM (P less than 0.01). The antilipolytic effects and the cyclic-AMP-lowering effects of the polyamines were almost completely prevented in the presence of different phosphodiesterase (PDE) inhibitors (3-isobutyl-1-methylxanthine and RO 20-1724) and, in addition, polyamines had no effect on lipolysis stimulated by dibutyryl cyclic AMP, indicating that polyamines may inhibit lipolysis by activating the PDE enzyme. This latter suggestion was confirmed by demonstrating that spermine (5 mM) significantly enhanced the low-Km PDE enzyme activity (P less than 0.01). Finally, the amounts of polyamines present in isolated adipocytes were measured, and the estimated cytoplasmic concentrations were 0.02 mM (putrescine), 0.86 mM (spermidine), and 1.0 mM (spermine). It is concluded that polyamines may possibly be involved in the physiological regulation of triacylglycerol mobilization in adipocytes.