AbstractRan, a Ras‐related GTPase, is required for transporting proteins in and out of the nucleus during interphase and for regulating the assembly of microtubules. cDNA cloning shows that rat testis, like mouse testis, expresses both somatic and testis‐specific forms of Ran–GTPase. The presence of a homologous testis‐specific form of Ran–GTPase in rodents implies that the Ran–GTPase pathway plays a significant role during sperm development. This suggestions is supported by distinct Ran–GTPase immunolocalization sites identified in developing spermatids. Confocal microscopy demonstrates that Ran–GTPase localizes in the nucleus of round spermatids and along the microtubules of the manchette in elongating spermatids. When the manchette disassembles, Ran–GTPase immunoreactivity is visualized in the centrosome region of maturing spermatids. The circumstantial observation that fractionated manchettes, containing copurified centrin‐immunoreactive centrosomes, can organize a three‐dimensional lattice in the presence of taxol and GTP, points to the role of Ran–GTPase and associated factors in microtubule nucleation as well as the potential nucleating function of spermatid centrosomes undergoing a reduction process. Electron microscopy demonstrates the presence in manchette preparations of spermatid centrosomes, recognized as such by their association with remnants of the implantation fossa, a dense plate observed only at the basal surface of developing spermatid and sperm nuclei. In addition, we have found importin β1 immunoreactivity in the nucleus of elongating spermatids, a finding that, together with the presence of Ran–GTPase in the nucleus of round spermatids and the manchette, suggest a potential role of Ran–GTPase machinery in nucleocytoplasmic transport. Our expression and localization analysis, correlated with functional observations in other cell systems, suggest that Ran–GTPase may be involved in both nucleocytoplasmic transport and microtubules assembly, two critical events during the development of functional sperm. In addition, the manchette‐to‐centrosome Ran–GTPase relocation, together with the similar redistribution of various proteins associated to the manchette, suggest the existence of an intramanchette molecular transport mechanism, which may share molecular analogies with intraflagellar transport. Mol. Reprod. Dev. 63: 131–140, 2002. © 2002 Wiley‐Liss, Inc.