In digitonin-permeabilized adipocyte suspensions, adenylate cyclase activation was analyzed by an in situ assay and compared to lipolytic activity which was determined under essentially identical assay conditions. This approach permitted direct comparison of the regulatory responses of these two processes. Adenylate cyclase was activated by isoproterenol in permeabilized adipocytes, but digitonin caused a decrease in sensitivity to the beta-agonist. On the other hand, activation of adenylate cyclase by forskolin and NaF was unaffected by digitonin. Inhibition of isoproterenol-activated adenylate cyclase by propranolol was also unaffected by digitonin. These observations are consistent with a partial uncoupling by digitonin of the interaction between occupied beta-adrenergic receptors and the stimulatory GTP-binding component (Ns). Despite permeabilization, adenylate cyclase activity retained the capability to activate the lipolytic pathway. Activation of adenylate cyclase and lipolysis by isoproterenol was first detectable at 10(-8) M. At 10(-7) M isoproterenol, when the cyclase activity increase was 11 pmol cAMP formed/min X 10(6) cells (21% increase over basal), lipolysis was increased 153%. Forskolin was a more effective activator of adenylate cyclase than isoproterenol, increasing activity maximally by greater than 7-fold in contrast to a maximum of 4.5-fold activation by the beta-agonist. Forskolin-dependent adenylate cyclase activity increased by 63 pmol cAMP/min X 10(6) cells, however, before an increase in lipolysis was first detected (33% increase). Thus, cAMP generated by the two lipolytic agents was not identical in its lipolytic potential. Maximum rates of lipolysis were equal for the two agents. Insulin (10(-9) M) exerted an antilipolytic effect on lipolysis in permeabilized adipocytes whether the lipolytic activator was isoproterenol or forskolin. At 10(-6) and 10(-4) M isoproterenol, the magnitudes of the antilipolytic effect were 35% and 21%, respectively, whereas forskolin-activated lipolysis (at 10(-6) M) was inhibited 22%. Insulin had no effect on adenylate cyclase activity stimulated by either agent and assayed under essentially identical conditions. In summary, direct comparison of adenylate cyclase activity and lipolysis in digitonin-permeabilized adipocytes demonstrated a more efficient coupling of adenylate cyclase to lipolysis when the enzyme was activated by isoproterenol rather than forskolin. Additionally, the antilipolytic effect of insulin was preserved in the permeabilized adipocytes, but this effect did not involve inhibition of adenylate cyclase.