A recently described family of "orphan" receptors, called Mas-related G-protein-coupled receptors (Mrg), is preferentially expressed in small nociceptive neurons of the rodent and human dorsal root ganglia (DRG). Mrg are activated by high affinity peptide fragments derived from the proenkephalin A gene, e.g. BAM22 (bovine adrenal medullary). To study the histological distribution and functional properties of these receptors, we combined confocal immunohistochemistry in rat DRG and dermis whole mounts, using new antibodies against the rat Mas-related G-protein-coupled receptor C (MrgC), with single-fiber recordings and neurochemical experiments using isolated hind-paw skin and sciatic nerve. In lumbar DRG we found cytoplasmic MrgC labeling mainly in small- and medium-sized neurons; coexpression with isolectin B4 (46%) and transient receptor potential vanilloid receptor 1 channel protein (TRPV1) (52%) occurred frequently, whereas calcitonin gene-related peptide (CGRP) was rarely colocalized with MrgC in DRG (11%) and dermal nerve fibers (6%). One of the MrgC agonists, BAM22, more than doubled the heat-induced cutaneous CGRP release from rat and mouse skin. The effect of BAM22, also known to activate opioid receptors, was further enhanced by combination with naloxone that had no effect on its own. This sensitizing effect proved to be independent of secondary prostaglandin formation, mast cell degranulation, protein kinase C (PKC) activation and independent of TRPV1. Nonetheless, the capsaicin-induced CGRP release was also sensitized. Receptive fields of 26 mechano-heat sensitive C-fibers were treated with MrgC agonists. Only one unit was strongly and repeatedly excited and showed a profound sensitization to heat upon BAM22+naloxone. Two other established MrgC agonists (gamma2-melanocyte stimulating hormone and BAM8-22) were ineffective. Thus, BAM22 sensitizes the capsaicin- and heat-induced CGRP release in an apparently MrgC-unrelated way. The sensitization to heat appears unusually resistant against pharmacological interventions and does not involve TRPV1.