Isocratic reverse phase analytical high performance liquid chromatography (HPLC) has been used to examine naturally occurring imidazoles of cardiac and skeletal muscles. Elution of muscle extracts with a phosphate buffer mobile phase from columns packed with hypersil ODS (5 micron) resulted in good separation of the skeletal muscle imidazole-containing dipeptides carnosine and anserine. Measured concentrations corresponded to published values. N-Acetyl forms that were not commercially available were prepared from their parent compounds and their identities verified by NMR-spectroscopy. Examination of frog cardiac muscle confirmed the presence of N-acetylhistidine and also indicated the presence of its 1-methyl derivative. Extracts of mammalian cardiac muscle were examined by HPLC which indicated the presence of low concentrations of carnosine but substantial amounts of N-acetyl forms of histidine, 1-methylhistidine, carnosine and anserine. Fractions corresponding to the numerous peaks were examined using staining systems specific for certain chemical features and compared to results obtained for commercial or synthetic standards. Results of these tests supported the chromatographic data. The total concentrations in cardiac muscle of these imidazole-containing substances (approx. 10 mM) is sufficient to alter significantly the sensitivity of their contractile apparatus to calcium ions.