The complete amino acid sequence of Tetrahymena pyriformis H 2 B histone was determined. The histone was obtained as described in the preceding paper [Nomoto, M. & Iwai, K. (1982) J. Biochem. 91, 719-723]. The purified histone was digested with an arginine-specific protease, clostripain, and the peptides, fragmented at 7 arginyl bonds and also at many of the 20 lysyl bonds, were fractionated by repeating column chromatography; most of these peptides were sequenced by Edman degradation. The chymotryptic peptides overlapping the clostripain peptides were obtained by limited or more extensive digestion of intact histone. The sequencing of these peptides led to reasonable aligning of the clostripain peptides. Thus, the sequence of 119 amino acid residues (mol. wt. 13, 316 for the unmodified form) has a completely α-N-blocked proline at residue 1 and a partially ε-N-acetylated lysine at residue 3. This sequence is compared with the known sequences of calf thymus and other H2B histones, and the implications for the structure and function relationship of this histone species and also for the phylogeny of protozoa are discussed.