Microtubules in the cytoplasm of rat Sertoli cell stage VI-VIII testicular seminiferous epithelium were studied morphometrically by electron microscopy. The Sertoli cell microtubules demonstrated axonal features, being largely parallel in orientation and predominantly spaced one to two microtubule diameters apart, suggesting the presence of microtubule-bound spacer molecules. Testis microtubule-associated proteins (MAPs) were isolated by a taxol, salt elution procedure. Testis MAPs promoted microtubule assembly, but to a lesser degree than brain MAPs. High molecular weight MAPs, similar in electrophoretic mobilities to brain MAP-1 and MAP-2, were prominent components of total testis MAPs, though no shared immunoreactivity was detected between testis and brain high molecular weight MAPs using both polyclonal and monoclonal antibodies. Unlike brain high molecular weight MAPs, testis high molecular weight MAPs were not heat stable. Testis MAP composition, studied on postnatal days 5, 10, 15, and 24 and in the adult, changed dramatically during ontogeny. However, the expression of the major testis high molecular weight MAP, called HMW-2, was constitutive and independent of the development of mature germ cells. The Sertoli cell origin of HMW-2 was confirmed by identifying this protein as the major MAP found in an enriched Sertoli cell preparation and in two rat models of testicular injury characterized by germ cell depletion. HMW-2 was selectively released from testis microtubules by ATP and co-purified by sucrose density gradient centrifugation with MAP-1C, a neuronal cytoplasmic dynein. The inhibition of the microtubule-activated ATPase activity of HMW-2 by vanadate and erythro-(2-hydroxy-3-nonyl)adenine and its proteolytic breakdown by vanadate-dependent UV photocleavage confirmed the dynein-like nature of HMW-2. As demonstrated by this study, the neuronal and Sertoli cell cytoskeletons share morphological, structural and functional properties.