Actin isolated from nearly every eukaryotic species contains approximately 1 mol 3-methylhistidine/mol protein. His73 in actin has been shown, by protein sequencing, to be the site of methylation. The methylation occurs enzymically and post-translationally. A rabbit skeletal muscle myofibrillary fraction has previously been shown to contain a histidine methyltransferase activity that is actin specific. Detailed study of this enzyme has been hampered by lack of a suitable substrate assay. Naturally occurring actins are poor substrates for the enzyme, presumably due to prexistent methylation at His73. In this study, two potential alternative substrates have been investigated. These are a chicken beta-actin expressed in Escherichia coli as a fusion protein with 80 amino acids of an influenza protein, NS1, and a synthetic peptide, Tyr-Pro-Ile-Glu-His-Gly-Ile-Ile-Thr, corresponding to residues 69-77 of actin. Both substrates were covalently methylated at histidine residues in the presence of S-adenosylmethionine and partially purified enzyme fractions from rabbit muscle. In methylation experiments employing the fusion actin in the form of inclusion bodies, 3-methylhistidine is the major product, as is the case when soluble muscle or non-muscle actin is used. However, for the synthetic peptide, the methylated product primarily contained 1-methylhistidine and only a small amount of the isomeric 3-methylhistidine. Further investigations revealed that the peptide was recognized by carnosine N-methyltransferase, another histidine methyltransferase found in muscle tissue. Carnosine N-methyltransferase appears to copurify with the actin-methylating enzyme in preliminary fractionation experiments. Separation of the two methyltransferase activities is described.