The influence of sympathetic innervation on the growth and intrinsic rate of beating established by fetal rat heart was studied by culturing fetal atrial tissue in sympathetically innervated and denervated anterior eye chambers of adult Sprague-Dawley rats. One anterior eye chamber in each host rat was sympathetically denervated by removing the ipsilateral superior cervical ganglion. In oculo, atrial grafts were vascularized by blood vessels sprouting from the iris and innervated by sympathetic and parasympathetic fibers from the ground plexus of the iris. Innervation was assessed by light-activated efferent nerve stimulation to the grafts that changed their rates of beating. The norepinephrine contents of 16 atria cultured for 2.5 months in sympathetically innervated and denervated eye chambers were 5.7 ± 1.1 ng/implant vs. 0.2 ± 0.07 ng/implant (mean ± SEM), indicating permanent sympathetic denervation of the anterior eye chamber and the implanted atria. By 8 weeks in oculo, atria maturing in sympathetically innervated anterior eye chambers were 86% larger than those in denervated eye chambers (2.22 ± 0.29 vs. 1.19 ± 0.13 mm2); the weight of innervated transplants was over 3 times that of noninnervated grafts (2.35 ± 0.75 vs. 0.76 ± 0.21 mg). After implanted atria had ceased growing rapidly (2.5 months in oculo), bipolar electrodes were implanted adjacent to the cornea to record impulses from atrial grafts while host rats were unanesthetized. The dark-adapted baseline heart rates of sympathetically innervated and noninnervated atria were virtually identical (289 vs. 290 bpm). Graft intrinsic heart rate was estimated by combined β-adrenergic and muscarinic receptor blockade with atenolol (1.0 mg/kg) and methylatropine (10 μg/kg). Sympathetically innervated transplants had lower intrinsic heart rates than noninnervated atria (134 ± 25 vs. 213 ± 12 bpm). These data suggest that sympathetic innervation of the developing heart influences both growth and intrinsic rate of beating.