The peptidergic innervation of proximal (internal diameter, > 0.8 mm) and distal (internal diameter, < 0.8 mm) regions of human epicardial coronary arteries was investigated by means of immunohistochemical, chromatographic, radioimmunological, and in vitro pharmacological techniques. The use of an antiserum to the general neuronal marker protein gene product 9.5 revealed that the proximal part of epicardial arteries possessed a relatively sparse supply of nerve fibers forming a loose network in the adventitia. The perivascular network increased in density as the vessels were followed distally. In both proximal and distal regions, the majority of nerve fibers possessed neuropeptide Y and tyrosine hydroxylase immunoreactivity. Calcitonin gene-related peptide (CGRP)- and substance P-immunoreactive nerve fibers were very sparse in the proximal region of the arteries and increased in number distally. Only a few scattered vasoactive intestinal peptide (VIP)-immunoreactive nerve fibers were detected in both arterial regions. The use of high-performance liquid chromatography and radioimmunoassay revealed that the immunoreactive material present in coronary artery extracts closely resembled synthetic peptides. An in vitro pharmacological method demonstrated that neuropeptide Y elicited no detectable response in either proximal or distal arterial segments. In contrast, CGRP, substance P, and VIP all produced a concentration-dependent relaxation of both arterial regions. CGRP and substance P were stronger and more potent than VIP. CGRP and substance P induced a more potent response in distal compared with proximal regions of the arteries. These results suggest that the peptidergic nerves supplying human large epicardial coronary arteries may be predominantly involved in mediating vasodilation.