A new S-II cDNA clone was isolated from a rat testis library. This cDNA contained an open reading frame encoding 299 amino acid residues. The deduced amino- and carboxyl-terminal regions were very similar with those of Ehrlich cell S-II, which we reported previously, but the sequence of the intervening 46 amino acid residues was unique. This new S-II was expressed in the testis but not in the other rat tissues examined, suggesting that it was a testis-specific S-II. Recombinant testis-specific S-II produced in Escherichia coli was shown to stimulate RNA polymerase II.