The major forms of cytochrome P-450 in the hepatic microsomes of rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (3MC) were isolated by sequential chromatography on n-octylamino-Sepharose 4B and DEAE-cellulose columns. These preparations exhibited single protein bands corresponding to cytochrome P-450s by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). High-performance liquid chromatography (HPLC) of these preparations on an anion-exchange column yielded three peaks from the PB-induced major cytochrome P-450 and a single peak from the 3MC-induced major cytochrome P-450. That the HPLC-isolated protein peaks were various forms of cytochrome P-450 was confirmed by spectral examination and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Examination of their absolute spectra revealed these cytochrome P-450s to be in a low-spin state. The lambda max of the reduced CO-complex spectra and molecular weights were found to be 450 nm and 53,000, respectively, for all the three HPLC-resolved cytochrome P-450s from PB-induced rats; and 448 nm and 56,000, respectively, for the HPLC-isolated cytochrome P-450 from 3MC-induced rats. The results demonstrate the effectiveness of HPLC in the determination of cytochrome P-450 multiplicity and charge heterogeneity.