Isocratic reverse-phase analytical HPLC has been used to examine naturally occurring imidazoles of rat brain. Elution of brain extracts with a phosphate buffer mobile phase from columns packed with Hypersil ODS (5 microns) resulted in good separation of the well-documented brain imidazole-containing dipeptides carnosine and homocarnosine. Measured concentrations corresponded to published values. Several further peaks observed had properties consistent with those of N-acetyl derivatives of compounds related to carnosine and homocarnosine. N-Acetyl forms not commercially available were prepared and their identities verified by nuclear magnetic resonance spectroscopy. A number of these had chromatographic properties identical to those of compounds in brain extracts. Fractions corresponding to some of the peaks were examined using staining systems specific for certain chemical features and compared with results obtained for commercial or synthetic standards. The results of these tests supported the chromatographic data. Thus, chromatographic and microchemical evidence is presented for the existence of N-acetyl forms of histidine, 1-methylhistidine, carnosine, anserine, and homocarnosine in rat brain.