During spermatogenesis, spermatids change position in the seminiferous epithelium along an axis that is perpendicular to the seminiferous tubule wall. During this period, spermatids are attached to apical invaginations of Sertoli cells. In areas of this attachment, unique junction plaques occur in Sertoli cells. These plaques consist of regions of the plasma membrane involved with intercellular adhesion, a layer of actin filaments that are hexagonally packed, and an underlying cistern of endoplasmic reticulum (ER). It previously has been proposed that these junction plaques, and therefore the attached spermatids, are translocated, by motor proteins, along microtubule tracts in the Sertoli cell. If this is true, microtubules should bind to the junction plaque in a nucleotide dependent fashion. To verify this prediction, seminiferous epithelia of the rat were separated from tubule walls and then mechanically fragmented. These epithelial preparations were incubated, in both the presence and absence of 10 mM Mg(+)(+)ATP, with exogenous microtubules stabilized with taxol. Then unbound microtubules were separated from microtubules bound to larger epithelial components by centrifuging the samples through a step sucrose gradient. The fraction enriched for elongate spermatids was collected and processed for electron microscopy. The results indicate that the junction plaques remain attached to spermatids, the plaques are intact, and the cytoplasmic face of the ER binds microtubules in a nucleotide dependent fashion. The results are consistent with the presence of motor proteins on the ER component of the junction plaques and with the general hypothesis of microtubule-dependent spermatid translocation.