The retina of anchovies is characterized by an unusual arrangement and ultrastructure of cones. In the retina of Japanese anchovies, Engraulis japonicus, three types of cones are distributed into rows. The nasal, central, temporal, and ventro-temporal regions of the retina were occupied exclusively by the long and short cones. Triple cones, made up of two lateral components and one smaller central component, were found only in the dorsal and ventro-nasal retinal regions. In the outer segments of all short and long cones from the ventro-temporal region, the lamellae were oriented along the cell axis and were perpendicular to the lamellae in the long cones, providing a morphological basis for the detection of polarization. This lamellar orientation is unique to all vertebrates. The cones were examined with respect to regional differentiation in their size and spectral properties via light microscopy, transmission electron microscopy, and microspectrophotometry. Various dimensions of cones were measured in preparations of isolated cells. The cones from the ventro-temporal region had different dimensions than cones of the same type located in other retinal regions. Triple cones from the dorsal region were significantly larger than triple cones from the ventro-nasal region. The spectral absorbance of the lateral components of triple cones in the ventro-nasal retina was identical to the absorbance of all long and short cones from the ventro-temporal region. These are shifted to shorter wavelengths relative to the absorbance of the lateral components of the triple cones located in the dorsal retina. Thus, the retina of the Japanese anchovy shows some features of regional specialization common in other fishes that improves spatial resolution for the upwards and forwards visual axis and provides spectral tuning in downwelling light environment. That results from the differentiation of cone types by size and by different spectral sensitivity of various retinal areas.