The effect of maturation on the contractile response to acetylcholine was studied in canine right atrial and right ventricular trabeculae. At any age the only inotropic response observed was negative. The decrease in contractile performance was always significantly greater in the atrial trabecula. Between 3 and 9 mo of age (12 dogs each group) there was a significant increase in the amount of negative inotropic action exerted by identical concentrations of acetylcholine. These data suggest that either the number of muscarinic receptors or the target cell response to acetylcholine increases during maturation.