Fetal and neonatal tissues are resistant to catecholamine-induced desensitization of essential physiological responses. We examined the mechanisms underlying the ontogeny of desensitization in neonatal rat heart for theβ-adrenergic receptor/adenylyl cyclase signaling cascade. Animals of different ages received isoproterenol daily for 4 days and cardiac membrane preparations were evaluated on the 5th day (6, 15, 25 days old and adults). Measurements were made of basal activity, activity stimulated by two agonists (isoproterenol or glucagon) that operate at different receptors but that share G_sas the transduction intermediate, or by forskolin-Mn²⁺to assess total catalytic capacity of the cyclase subunit; we also assessed inhibition of activity by carbachol which acts via muscarinic cholinergic receptors and G_i. Adult rats exhibited robust desensitization of the adenylyl cyclase response but the effect was heterologous in that equivalent loss of activity was seen for basal, isoproterenol-, and glucagon-stimulated activity; forskolin-Mn²⁺-stimulated activity was also decreased. Two factors contributed to desensitization: generalized reduction in membrane protein concentrations caused by cell enlargement (reduced surface-to-volume ratio), and specific interference with the G-protein component that couples receptors to the cyclase. Thus, after adjustment for changes in membrane protein, the desensitization of the forskolin-Mn²⁺response was no longer evident, but the effects on the other measures were still present. In addition, isoproterenol treatment produced crosstalk with the carbachol/G_isignaling pathway, with significant reductions in the ability of carbachol to inhibit adenylyl cyclase activity. Heterologous desensitization by isoproterenol was also present in 15 and 25 day old rats, but involved only selective components of the effects seen in adults. At 25 days, uncoupling of signals operating through G_sand G_iwas obtained without a reduction in forskolin-Mn²⁺-stimulated activity. At 15 days, only the effect on G_scoupling was seen. At 6 days, agonist-induced desensitization was not detectable and instead, heterologous sensitization was found. In these youngest animals, isoproterenol treatment produced a parallel increase in basal, isoproterenol-, glucagon- and forskolin-Mn²⁺-stimulated activity, unaccompanied by changes in membrane protein concentrations, indicating an increase in adenylyl cyclase catalytic activity. These results indicate that the ability to elicit desensitization is not an inherent property of cardiac cells but rather is acquired in distinct stages during development. Sensitization by agonists early in development may be important in preserving physiological responsiveness during ontogenetic surges of adrenergic activity.