Adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] was determined in vitro in fetal rabbit myocardial membranes from individual fetal pups at 21 to 31 days gestation (term, 31 days). Basal and NaF-stimulated adenylate cyclase activities did not change during 21-31 days gestation. Significant stimulation of the enzyme by l-isoproterenol was observed only in the presence of guanosine triphosphate (100 µM). Under these conditions, maximal adenylate cyclase stimulation by lisoproterenol (100 µM) was significantly higher at 25–31 than at 21 days gestation. Moreover, EC50(Kact) for l-isoproterenol at 25–31 days was significantly lower than at 21 days gestation. We conclude that, in fetal rabbit myocardial membranes, there is an increase in the sensitivity of adenylate cyclase stimulation by l-isoproterenol from 21 to 25–31 days gestation