The role of phosphodiesterase in glucagon resistance of large adipocytes was investigated. A comparison was made of phosphodiesterase activities of homogenates prepared from isolated small (mean diameter approximately 45 micro m) and large (mean diameter approximately 78 micro m) adipocytes, using various concentrations (5 x 10(-4) to 1 x 10(-7) M) of 3',5'-cAMP. Kinetic analyses revealed two distinct catalytic activities (high and low affinities) in both cell types; however, the activities of both high- and low-affinity enzymes were significantly elevated in large adipocytes. Lipolysis was measured in isolated adipocytes in the presence of different concentrations (0.1-0.6 mM) of the phosphodiesterase inhibitor aminophylline. Large adipocytes were less responsive to low levels of methylxanthine, suggesting that greater amounts of phosphodiesterase must be inhibited before lipolysis can be stimulated. To evaluate the influence of phosphodiesterase during glucagon-stimulated lipolysis, small and large adipocytes were incubated with a maximally effective concentration of glucagon (1.5 x 10(-6) M) in combination with various concentrations (0.1-0.6 mM) of aminophylline. Although the glucagon effect was potentiated in both cell types, the maximum lipolytic response of large adipocytes (at 0.4 mM aminophylline) was approximately 36% lower than that observed in small adipocytes (at 0.2 mM aminophylline). This reduction correlates closely with the decreased glucagon binding present in large cells; therefore, it appears that the glucagon-resistant state is adequately explained by elevations in phosphodiesterase levels and diminished glucagon-cell association.