Nestin is an intermediate filament (IF) protein (IFP) which occurs during early developmental stages and during regenerative processes in muscle and neuronal cells. The spatial and temporal localization of nestin in the developing testis of rat and mouse was studied by immunolabeling light and electron microscopy and by immunoblotting. Nestin localization was related to the localization of the other major IFPs specific for this tissue, i.e. cytokeratins, vimentin and desmin. Laminin immunocytochemistry and conventional microscopy were used to identify tissues and cells. With the incipient differentiation of the gonadal anlage, the reaction for nestin was weak in the gonadal ridge, whereas the cells of the mesonephric mesenchyme showed a prominent reaction for this IFP. The nestin-specific reaction in the epithelial mesonephric duct and tubules was weak and disappeared at an early phase of differentiation. With the development of the testis proper, nestin was transiently found in several cell types. Nestin was found as well as vimentin and cytokeratins in the Sertoli cells. In the interstitial cells nestin was found together with vimentin and desmin IFPs, and was most prominent in the differentiating myoid cells. After birth, nestin gradually disappeared from the testicular cells and in the rat at puberty was found only in the endothelial cells of some blood vessels. The abolished nestin synthesis in the testis was confirmed by immunoblotting. These results suggest that nestin is required transiently during the development of the testis and mesonephros. The temporary presence of nestin, and several other IFPs during these phases, coincides with key phases of urogenital sex differentiation. This may imply that the orchestrated synthesis of the IFPs nestin, cytokeratins, vimentin and desmin is likely to be linked with the genes regulating sex differentiation.