This paper reports on the presence of the conus arteriosus in the heart of the adult gilthead seabream, Sparus auratus (Perciformes, Teleostei). The junctional region between the single ventricle and the bulbus arteriosus has been studied by conventional light microscopy, and by scanning and transmission electron microscopy. In addition, fluorescent phalloidin and antibodies against the muscle myosin heavy chains, laminin and collagen type IV have been used. The conus arteriosus is a distinct muscular segment interposed between the ventricle and the bulbus arteriosus. It is clearly different from the bulbus arteriosus due to its myocardial nature. It can also be distinguished from the ventricular myocardium because: (1) it has a conus shape; (2) it is formed by compact, well-vascularized myocardium; (3) it is surrounded on its inner and outer faces by fibrous layers rich in collagen and elastin; (4) it constitutes the anatomical support of the so-termed conus valves; (5) it shows intense staining for laminin and type-IV collagen; and (6) the myocardial cells located close to the inner fibrous layer are helicoidally arranged. By contrast, the ventricular myocardium is highly trabecular, lacks a compacta, shows no vessels, and presents barely detectable amounts of laminin and collagen type IV. The presence of a distinct conus arteriosus in the heart of an evolutionary advanced teleost species indicates that the conus is not a vestigial segment from the evolutionary or embryological points of view. The characteristic spatial arrangement of the conus myocytes strongly suggests that the conus is implicated in the mechanical performance of the conus valves.