The amino acid sequence of human spleen histone H3 was investigated as a study in histone evolution, following previous investigations of human spleen histone H2B [Ohe, Y., Hayashi, H., & Iwai, K. (1979) J. Biochem. 85, 615-624] and H2A [Hayashi, T., Ohe, Y., Hayashi, H., & Iwai, K. (1980) J. Biochem. 88, 27-34]. The H3 fraction was obtained as described previously and further purified by Bio-Gel P-10 chromatography. The reduced and carboxymethylated H3 was digested with an arginine-specific protease, Clostripain, and the peptides were fractionated by repeated column chromatographies with reasonable recoveries. Most of these peptides, together with two tryptic peptides and one chymotryptic peptide obtained similarly, were sequenced by Edman degradation. Thus, the human H3 was deduced to have a sequence of 135 amino acid residues identical with that of the main component of calf thymus H3, except for one complete substitution (Ser for Cys-96) and four partial substitutions (Ser for Ala-31, Ala for Ser-87, Ile for Val-89, and gly for Met-90), which suggest the presence of at least two variants. Three methylated lysines (residues 9, 27, and 36) and two acetylated lysines (residues 14 and 23) were also found. The substitutions and modifications found in the human H3 are compared with those in the known H3 sequences of other eukaryotes, and the implications are discussed.