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The distribution of nerves immunoreactive to protein gene product 9.5 (PGP 9.5), tyrosine hydroxylase (TH), neuropeptide Y (NPY), substance P (SP) and calcitonin gene related peptide (CGRP) antisera was investigated in the atrioventricular valves of the Sprague-Dawley rat and the Dunkin-Hartley guinea pig using confocal and epifluoresence microscopy. No major differences were noted between the innervation of the mitral and tricuspid valves in either species. For all antisera the staining was more extensive in the guinea pig valves. Two distinct nerve plexuses separated by a 'nearly nerve free' zone were identified in both species with each antiserum tested. This was most apparent on the anterior cusp of the mitral valve. The major nerve plexus extends from the atrioventricular ring through the basal, intermediate and distal zones of the valves towards the free edge of the valve cusp. These nerve bundles, arranged as primary, secondary and tertiary components, ramify to the free edge of the valve and extend to the attachment of the chordae. They do not contribute to the innervation of the chordae tendineae. The second, minor chordal plexus, runs from the papillary muscles through the chordae tendineae and passes parallel to the free edge of the cusp. The nerves of this minor plexus are interchordal, branching to terminate mainly in the distal zone, free edge of the valve cusp and adjacent chordae tendineae. Some interchordal nerve fibres loop from a papillary muscle up through a chorda, along the free edge and pass down an adjacent chorda into another papillary muscle. The nerve fibres of the major and minor plexuses intermingle although no evidence was found for interconnectivity between them. In the distal zone between the major plexus which extends from the base of the valve and the minor chordal plexus there is a zone completely free of nerves staining with antisera to TH and NPY. Occasional nerves which stained positive for PGP 9.5, SP and CGRP immunoreactivities crossed this 'nearly nerve free zone' passing either from the chordal/free edge nerves to the intermediate and basal zones or vice versa. An additional small nerve plexus which displayed immunoreactivity to CGRP antiserum extended from the atrioventricular ring into the basal zone of the valve cusp. Not all chordae tendineae displayed immunoreactive nerve fibres. It is concluded that the innervation patterns of the sensory and sympathetic neurotransmitters and neuropeptides examined in the atrioventricular valves of the rat and guinea pig are ubiquitous in nature. The complexity of the terminal innervation network of the mammalian atrioventricular valves and chordae tendineae may contribute to the complex functioning of these valves in the cardiac cycle.