Bundles of microtubules occur adjacent to ectoplasmic specializations (ESs) that line Sertoli cell crypts and support developing spermatids. These microtubules are oriented parallel to the direction of spermatid movement during spermatogenesis. We propose a model in which ESs function as vehicles, and microtubules as tracks, for microtubule-based transport of spermatids through the seminiferous epithelium. Microtubule polarity provides the basis for the direction of force generation by available mechanoenzymes. As part of a more general study designed to investigate the potential role of microtubule-based transport during spermatogenesis, we have studied the polarity of cytoplasmic microtubules of Sertoli cells. Rat testis blocks were incubated in a lysis/decoration buffer, with and without exogenous purified bovine brain tubulin. This treatment results in the decoration of endogenous microtubules with curved tubulin protofilament sheets (seen as hooks in cross section). The direction of curvature of the hooks indicates microtubule polarity; that is, clockwise hooks are seen when viewing microtubules from the plus to the minus end. We found that, in Sertoli cells, most of the hooks were orientated in the same direction. Significantly, when viewed from the base of the epithelium, hooks pointed in a clockwise direction. The clockwise direction of dynein arms on axonemes of sperm tails, in the same section, provided an internal check of the section orientation. Electron micrographs of fields of seminiferous epithelium were assembled into montages for quantitative analysis of microtubule polarity. Our data indicate that Sertoli cell cytoplasmic microtubules are of uniform polarity and are orientated with their minus ends toward the cell periphery. These observations have significant implications for our proposed model of microtubule-based transport of spermatids through the seminiferous epithelium.