During spermiogenesis, the shape of the spermatid nucleus, which is spherical, changes and it becomes the sperm head. A microtubular structure called a manchette is thought to be involved in this morphogenetic process. In this report, we demonstrate the localization of cytoplasmic dynein and manchette development by a double immunofluorescence technique using anti-bovine brain MAP 1C and anti-tubulin. Before step 6 of the Leblond and Clermont staging, the microtubules showed a fine reticular network, and the dynein staining was homogeneous. In step 6, the microtubular network was concentrated around the nucleus. The manchette developed in step 7 spermatids, and was fully formed, with a skirt-like appearance, covering the nuclear surface in step 8. Dynein fluorescence was associated with the microtubular manchette in steps 7-10. During these steps, the nucleus was protruded from the cytoplasm. In steps 11-13, the most active stages in nuclear shaping, the dynein was densely localized at the nuclear surface covered by the manchette. As the nucleus acquired a shape similar to the mature spermatozoon at step 14, the dynein fluorescence was localized only at the concave side of the nuclear caudal edge. The manchette became narrower and elongated. In step 15, the manchette extended into the elongated cytoplasm, diminishing during steps 16-18. The localization of the dynein was limited to the ventral aspect of the caudal head in these steps. There was little dynein fluorescence in mature spermatozoa. Immunoelectron microscopy showed positive reactions in the nuclear envelope and the inner region of the microtubular manchette. These observations suggest that cytoplasmic dynein, possibly bound to the nuclear envelope, and manchette microtubules are involved in the protrusion of the spermatid nucleus from the cytoplasm.