Following elongation of spermatids in mammals, the histones are replaced by a set of basic nuclear transition proteins; in the rat there are four, named TP1-TP4. Of these, TP1 and TP2 are well characterized. Here we report the purification to homogeneity of TP4 from rat spermatids. It is a low molecular mass (about 13-20 kDa) basic protein with arginine and lysine constituting 24 mol % and histidine 2.2 mol %. Its 25 N-terminal amino acids were sequenced, and no sequence homologies with any known protein were found. Polyclonal antibodies raised against it in rabbit did not cross-react with other transition proteins, protamines, or histones. The presence of TP4 during sperm development was monitored by cell separation studies. No TP4 was detected in round spermatids, and along with TP1 and TP2, it is present in step 13-15 spermatids and its amount decreased in steps 16-19. Trace amounts of TP4 were also detected in epididymal sperm. A possible role for TP4 in spermatid and sperm chromatin structure is discussed.