The previous study showed that the H2A histone of the protozoan Tetrahymena pyriformis comprises equimolar amounts of two variants, H2A(1) and H2A(2), and their sequences of 137 and 132 residues, respectively, are blocked at the N-terminal and possibly partially modified in the homogeneous N-terminal and heterogeneous C-terminal regions [Fusauchi, Y. & Iwai, K. (1983) J. Biochem. 93, 1487-1497]. Now, the N-terminal blocking group was identified as alpha-N-acetyl by 1H NMR spectroscopy of the N-terminal blocked serine residue isolated by carboxypeptidase digestion of the N-terminal tryptic peptide (residues 1-5). Two epsilon-N-acetylated (16 and 14%) lysine residues positioned at 5 and 12, respectively, were found on carboxypeptidase digestion and subsequent amino acid analysis of a blocked N-terminal tryptic peptide (res. 1-8) and on aminopeptidase digestion analysis of another tryptic peptide (res. 11-17), both containing two lysine residues. The C-terminal BrCN fragment (res. 119-137) of H2A(1) containing three, two, one, and no phosphoserine, and that (res. 119-132) of H2A(2) containing two, one, and no phosphoserine were isolated by ion-exchange chromatography and determined by carboxypeptidase digestion analyses. Similar analyses of the tryptic peptides derived from these BrCN fragments showed that H2A(1) was phosphorylated at serine residues 122 (76%), 124 (15%), and 129 (25%); and H2A(2) at serine residues 122 (81%) and 128 (44%). This is the first time that C-terminal phosphorylation has been found in nucleosome-core histones.