The central projections and neurochemistry of vagal afferent neurones supplying the heart in the rat were investigated by injecting cholera toxin B-subunit into the pericardium. Transganglionically transported cholera toxin B-subunit was visualized in the medulla oblongata in axons and varicosities that were predominantly aggregated in the dorsomedial, dorsolateral, ventrolateral and commissural subnuclei of the caudal nucleus of the solitary tract. Unilateral vagal section in control rats prevented cholera toxin B-subunit labeling on the ipsilateral side of the nucleus of the solitary tract. Fluorescent and electron microscopic dual labeling showed colocalization of immunoreactivity for vesicular glutamate transporter 1, but only rarely vesicular glutamate transporters 2 or 3 with cholera toxin B-subunit in terminals in nucleus of the solitary tract, suggesting that cardiac vagal axons release glutamate as a neurotransmitter. In contrast, populations of vagal afferent fibers labeled by injection of cholera toxin B-subunit, tetra-methylrhodamine dextran or biotin dextran amine into the aortic nerve, stomach or nodose ganglion colocalized vesicular glutamate transporter 2 more frequently than vesicular glutamate transporter 1. The presence of other neurochemical markers of primary afferent neurones was examined in nucleus of the solitary tract axons and nodose ganglion cells labeled by pericardial cholera toxin B-subunit injections. Immunoreactivity for a 200-kDa neurofilament protein in many large, cholera toxin B-subunit-labeled nodose ganglion cells indicated that the cardiac afferent fibers labeled are mostly myelinated, whereas binding of Griffonia simplicifolia isolectin B4 to fewer small cholera toxin B-subunit-labeled ganglion cells suggested that tracer was also taken up by some non-myelinated axons. A few labeled nucleus of the solitary tract axons and ganglion cells were positive for substance P and calcitonin gene-related peptide, which are considered as peptide markers of nociceptive afferent neurones. These data suggest that the population of cardiac vagal afferents labeled by pericardial cholera toxin B-subunit injection is neurochemically varied, which may be related to a functional heterogeneity of baroreceptive, chemoreceptive and nociceptive afferent fibers. A high proportion of cardiac neurones appear to be glutamatergic, but differ from other vagal afferents in expressing vesicular glutamate transporter 1.