Adenosine functions as a counterregulatory hormone in the myocardium by decreasing work and thereby protecting the myocardium against ischemia. Functional adenosine A1 receptors could serve as an important regulatory system in the developing preinnervated heart by balancing the humoral sympathetic input to the heart. The aims of this study were to determine if A1 adenosine receptors were functionally coupled to their Gi protein in the immature preinnervated heart and to determine if A1 adenosine receptors were present in greater numbers in the immature heart. One- to 3-day-old rat ventricular cardiomyocyte cultures were exposed to (1) control conditions; (2) isoproterenol, a beta-receptor agonist, (3) R-PIA, an A1 agonist, or (4) isoproterenol and R-PIA, cAMP levels were determined by RIA in each group. Adenosine A1 receptor density and the equilibrium dissociation constant were determined by binding of an adenosine A1 receptor antagonist in newborn, 1-week-old, 2-week-old, and adult rat hearts. A1 stimulation decreased the isoproterenol-induced increase in cAMP by 30%, demonstrating functional A1 receptors in immature preinnervated myocytes. The A1 receptor density in the newborn age group was twice the adult and 2-week-old level. We conclude that A1 receptors in the immature heart are functionally coupled to their effector and that A1 receptors are present in greater numbers in the immature heart.