A partial amino-acid sequence of Tetrahymena histone H4 has been determined and differs significantly from the sequence of calf or pea H4. The amino terminus of Tetrahymena H4, unlike that of other H4s so far examined, is not acetylated. Of 66 residues determined, one is a single-residue insertion, one a single-residue deletion, and thirteen are amino-acid replacements with respect to the calf thymus H4 sequence. Most of the amino-acid replacements are nonconservative and are distributed nonrandomly, with a strong concentration in the amino-terminal arm. The first four lysines are partially acetylated. All but two of the replacements can be explained by single nucleotide substitutions at the level of the gene. The similarity in sequence of calf and pea H4 coupled with the substantial differences displayed by Tetrahymena H4 suggest that the divergence of protozoa and higher eukaryotes substantially antedates the divergence of plants and animals. Furthermore, quantitative analysis of the data requires either that the rate of H4 evolution be considerably more rapid than previously thought or that the rate be different for different periods of evolution or for different lines of descent.