Intrinsic cardiac neurons are core elements of a complex neural network that serves as an important integrative center for regulation of cardiac function. While mouse models are used frequently in cardiovascular research, very little is known about mouse intrinsic cardiac neurons. Accordingly, we have dissociated neurons from adult mouse heart, maintained these cells in culture, and defined their basic phenotypic properties. Neurons in culture were primarily unipolar, and 89% had prominent neurite outgrowth after 3 days (longest neurite length of 258 ± 20 µm, n=140). Many neurites formed close appositions with other neurons and non-neuronal cells. Neurite outgrowth was drastically reduced when neurons were kept in culture with a majority of non-neural cells eliminated. This finding suggests that non-neuronal cells release molecules that support neurite outgrowth. All neurons in coculture showed immunoreactivity for a full complement of cholinergic markers but about 21% also stained for tyrosine hydroxylase, as observed previously in sections of intrinsic cardiac ganglia from mice and humans. Whole-cell patch clamp recordings demonstrated that these neurons have voltage-activated sodium current that is blocked by tetrodotoxin, and that neurons exhibit phasic or accommodating patterns of action potential firing during a depolarizing current pulse. Several neurons exhibited a fast inward current mediated by nicotinic ACh receptors. Collectively, this work shows that neurons from adult mouse heart can be maintained in culture and exhibit appropriate phenotypic properties. Accordingly, these cultures provide a viable model for evaluating the physiology, pharmacology, and trophic factor sensitivity of adult mouse cardiac parasympathetic neurons.