1. The negative chronotropic effects of acetylcholine and carbachol on isolated rat right atria were examined at 0, 4, 8, and 16 weeks after birth. 2. Acetylcholine produced negative chronotropic responses at all ages and completely abolished spontaneous beating at its maximum effective concentration. 3. The sensitivity to acetylcholine, expressed in terms of ED50 values, was higher at 0 and 4 weeks than at 8 and 16 weeks, ED50 values (microM) at 0, 4, 8 and 16 weeks being 9.5 +/- 1.8 (n = 12), 13.2 +/- 3.4 (n = 11), 59.3 +/- 10.9 (n = 14) and 51.5 +/- 17.5 (n = 5), respectively. 4. Neostigmine produced a leftward shift of the concentration-response curve for acetylcholine both at 4 and 8 weeks after birth. The shift was larger at 8 weeks and no difference in sensitivity to acetylcholine was observed between the two ages in the presence of neostigmine. 5. Further, no developmental changes were observed in the sensitivity to carbachol, which is not hydrolyzed by cholinesterase. 6. We concluded that the chronotropic sensitivity to acetylcholine of rat atria decreases post-natally during the period between 4 and 8 weeks after birth due to increase in cholinesterase activity.