The relationship between biological maturation and adenylate cyclase activity was studied in membrane preparations of rabbit ventricular muscle. Basal adenylate cyclase activity was lower in the adult than in the 1-day-old neonate or 27-day-old fetus. Maximal stimulation of adenylate cyclase by isoproterenol was 2.5 times greater and the EC50 values were 2-fold higher in the adult than the 27-day-old fetus or 1-, 7- and 12-day-old neonate. No significant differences in isoproterenol- or Mg++-stimulated activity were observed among the younger age groups nor was the Mg++-stimulated Vmax of adenylate cyclase significantly affected by biological maturation. Sodium fluoride, guanyl-5'yl imidodiphosphate and GTP also stimulated adenylate cyclase activity in a dose-dependent fashion similar to isoproterenol. Sodium fluoride (2.5-10 mM) increased adenylate cyclase activity in the adult to a significantly greater extent than the 1-day-old neonate. Guanyl-5'yl imidodiphosphate and GTP (0.1-10.0 microM) augmented adenylate cyclase activity to approximately the same degree (although some small differences were observed) in the fetus, neonate and adult. However, when guanyl-5'yl imidodiphosphate was preincubated with membrane preparations before in vitro assay, adenylate cyclase activity was increased 10-fold in the adult, whereas membranes from 1-day-old animals were unaffected. These data suggest that the processes regulating hormonal and pharmacological activation of adenylate cyclase are modified during biological maturation.