Postnatal development of autonomic control of heart rate was evaluated in the sinus nodes of isolated, perfused right atria obtained from 21 sibling puppies in 3 different litters between 3 and 11 wk postpartum. Age-related changes in response to autonomic blockade indicated that propranolol administered after atropine had its most profound direct depressive effect on less mature atrial cells. In the youngest sinus nodes the familiar antimuscarinic action (increase in sinus rate) of atropine was regularly preceded by a brief period of cholinomimetic action (a marked slowing of sinus rate) in the youngest sinus nodes. These two opposing effects of atropine underwent a developmental change during the 2-mth study. The cholinomimetic action diminished, whereas the antimuscarinic action increased as a function of age.