Apart from the well-studied melanocytes of the skin, eye and inner ear, another population has recently been described in the heart. In this study, we tracked cardiac melanoblasts using in situ hybridization with a dopachrome tautomerase (Dct) probe and Dct-LacZ transgenic mice. Large numbers of melanoblasts were found in the atrioventricular (AV) endocardial cushions at embryonic day (E) 14.5 and persisted in the AV valves into adulthood. The earliest time Dct-LacZ-positive cells were observed in the AV endocardial cushions was E12.5. Prior to that, between E10.5 and E11.5, small numbers of melanoblasts traveled between the post-otic area and third somite along the anterior and common cardinal veins and branchial arch arteries with other neural crest cells expressing CRABPI. Cardiac melanocytes were not found in the spotting mutants Ednrb s-l/s-l and Kit w-v/w-v, while large numbers were observed in transgenic mice that overexpress endothelin 3. These results indicate that cardiac melanocytes depend on the same signaling molecules known to be required for proper skin melanocyte development and may originate from the same precursor population. Cardiac melanocytes were not found in zebrafish or frog but were present in quail suggesting an association between cardiac melanocytes and four-chambered hearts.