Adenosine acts via specific receptors (A1, A2a, A2b, and A3) to exert potent biological effects. Although postnatal influences of adenosine on the heart are recognized, little is known about the influence of adenosine on fetal cardiac function. Recently, we have discovered that A1 receptors are expressed in cardiac tissue at very early stages of development(Devel. Brain Res. 89:202, 1995). We now aim to determine if adenosine influences cardiac function at embryonic stages. Cultures ofin vitro differentiated cardiac tissue from mice at post-conception(PC) 9.5 were established. At 12 hrs after cells were cultured, spontaneously contracting cardiac tissue was observed (100 - 160 bpm). Treatment of cultures with the A1 receptor agonist N6-cyclopentyladenosine (CPA), resulted in dose dependent decreases in cardiac rates (Ki = 48 nM; n = 6) and completely arrested cardiac contractions at 1 uM. This effect was reversed by the A1 receptor antagonist 8-phenyl-1,3-dipropylxanthine (CPX; Ki = 26 nM; n = 3); CPA action also was blocked by pretreatment with pertussis toxin. In contrast to CPA, concentrations of drugs that activate A2 or A3 receptors, did not alter heart rates. Treatment with the beta-receptor agonist, isoproterenol, resulted in 30-40% increases in heart rates. This effect was completely blocked by CPA. Receptor autoradiography using [3H]CPX demonstrated A1 receptor expression in the cardiac cultures. These data now identify A1 adenosine receptors as potent regulators of embryonic cardiac activity.