Transition protein 2 (TP2) of the rat was isolated by differential precipitation with trichloroacetic acid, chromatography over Bio-Rex 70, and preparative gel electrophoresis. A polyclonal rabbit antiserum was raised that did not cross-react with unrelated acid-soluble proteins from liver or testes. The antiserum was used to identify TP2-related proteins obtained from testes of mice, hamsters, guinea pigs, rabbits, and boars by Western blotting. Immunohistochemical techniques were used to localize TP2 in paraffin-embedded testis sections from mice and rats. In both species, TP2 was first detected in spermatids that had essentially completed the morphological change from a round to an elongate nucleus and that were undergoing chromosomal condensation (spermatids of step 13 in rat and step 12 in mouse). TP2 was retained in spermatid nuclei until early step 16 in the rat and step 14 in the mouse. Serial sections of rat testis exposed separately to antisera to TP1 and TP2 showed that the great majority of labeled tubules were reactive to both antisera. However, in occasional tubules, TP1 reactivity was retained in relatively late spermatids that were negative for TP2. Thus both TP1 and TP2 appear in the nucleus essentially simultaneously, in association with the beginning of chromatin condensation and at a point well after much of the nuclear shaping has occurred.