The cytosolic enzymes extracted from rat hepatocytes were separated by native porous gradient-PAGE (PG-PAGE) and were detected with a sensitive and fast chemiluminescence (CL) imaging method. Several peroxidases including glutathione peroxidase, Cu/Zn-superoxidase dismutase, and some other metallo-enzymes such as catalase, carbonic anhydrase-III (CA-III) present in the cytosol of rat hepatocytes have been selectively and sensitively detected by the direct CL imaging method using the luminol-H2O2 chemiluminescent reagents. All detections after PG-PAGE were completed within 9 min. The linear range for the typical metallo-enzyme, e.g., CA-III is 0.754.9 mu g/mL, with a detection limit of 0.25 mu g/mL. In comparison with the traditional CBB-R250 staining method, the detection period decreased about 70 times and the detection sensitivity improved over ten times. Furthermore, two enzymes present in rat liver cytosol were identified employing MALDI-MS analysis of the tryptic digest after PG-PAGE.