The interphotoreceptor matrix (IPM) is a mixture of proteoglycans and glycoproteins through which metabolites must pass in transit between the retinal pigment epithelium (RPE) and the photoreceptor cells. In order to localize various species of chondroitin sulfates in the IPM of the normal and dystrophic rat retina, we have used monoclonal antibodies directed against 6-sulfated chondroitin sulfate (6S), 4-sulfated chondroitin sulfate (4S) and unsulfated chondroitin (OS). Immunofluorescence and immunoperoxidase methods were carried out on frozen and wax-embedded sections of rat eyes. In the normal rat retina, strong labeling with the 6S antibody was observed in the basal IS/OS zone and, to a lesser extent, in the photoreceptor interstices extending to the apical RPE surface. In the RCS retina, the IPM in the basal IS/OS zone and much of the outer segment debris zone were labeled intensely with 6S antibody, but no strong labeling was present at the apical RPE surface. The labeling pattern with OS antibody was similar to that of the 6S antibody for both normal and RCS retinas. We failed to detect any 4S antibody labeling of the IPM in both the normal and RCS retinas using these methods. These results indicate that in the rat retina, the IPM contains 6-sulfated chondroitin sulfate and unsulfated chondroitin proteoglycans that are most concentrated in the basal IS/OS zone and, to a lesser degree, between the photoreceptor outer segments. Furthermore, it appears that the rat IPM contains little or no 4-sulfated chondroitin sulfate or dermatan sulfate proteoglycan.