Small GTP-binding protein rab8 regulates transport from the TGN to the basolateral plasma membrane in epithelial cells and to the dendritic plasma membrane in cultured hippocampal neurons. In our approach to identify proteins involved in rhodopsin transport and sorting in retinal photoreceptors, we have found, using [32P]GTP overlays of 2D gel blots, that six small GTP-binding proteins are tightly bound to the post-Golgi membranes immunoisolated with a mAb to the cytoplasmic domain of frog rhodopsin. We report here that one of these proteins is rab8. About 50% of photoreceptor rab8 is membrane associated and approximately 13% is tightly bound to the post-Golgi vesicles. By confocal microscopy, antibody to rab8 specifically labels calycal processes and the actin bundles of the photoreceptor inner segment that extend inward to the junctional complexes that comprise the outer limiting membrane. Anti-rab8 shows a striking periodicity of high density labeling at 1 +/- 0.12 microns intervals along the actin bundles. Rhodopsin-bearing post-Golgi membranes cluster around the base of the cilium where rab8 and actin are also co-localized, as revealed by confocal microscopy of retinal sections double labeled with anti-rab8 and phalloidin. Microfilaments have been implicated in rod outer segment (ROS) disk morphogenesis. Our data suggest that rab6, which we have previously localized to the post-Golgi compartment, and rab8 associate with the post-Golgi membranes sequentially at different stages of transport. rab8 may mediate later steps that involve interaction of transport membranes with actin filaments and may participate in microfilament-dependent ROS disk morphogenesis.