Members of the GDNF family, which are important during peripheral nervous system development and kidney organogenesis, signal via Ret and GFRalpha receptors. Here we have studied their possible role in heart development. Gfra1 was expressed in the endocardial cushion mesenchyme at E12 and later, in the developing and mature valves, and in the walls of the aorta and the pulmonary trunk. Gfra2 was expressed in the outer layers of the aorta and pulmonary trunk and in the valves at E18-P60. Endocardial cells showed moderate Gfra2 mRNA and protein expression between E12 and E15. Gfra3 mRNA was detected, mainly postnatally, in scattered cells of the atria and the great vessels. In embryonic and postnatal rat cardiac ganglia, Ret and Gfra2 transcripts were seen in the neurons, whereas Gfra1 and Gfra3 mRNA were preferentially found in non-neuronal cells within the ganglia. GFRalpha2 immunoreactivity was seen in both cardiac ganglion neurons and their nerve fibers. There were no obvious non-neuronal defects in hearts of Ret-, GFRalpha1-, or GFRalpha2-deficient mice, suggesting that these receptors are not essential for gross cardiac development. However, E18 Ret-deficient mice exhibited a reduced volume of cardiac ganglia and cholinergic innervation of the ventricular conduction system. Moreover, adult Gfra2(-/-) mice showed reduced cholinergic innervation by 40% in their ventricles and by 60% in the ventricular conduction system. These findings indicate that GFRalpha2/Ret signaling is required for normal cholinergic innervation of heart.