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 G_i 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 β-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 At 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.