On-line capillary zone electrophoresis/electrospray ionization mass spectrometry (CZE-ESMS) and capillary isoelectric focusing/electrospray ionization mass spectrometry (CIEF/ESMS) were employed for protein analysis. The separation mechanisms and the detection limits of CZE/ESMS and CIEF/ESMS were compared by using model proteins including cytochrome c (horse heart), ribonuclease A (bovine pancreas), myoglobin (horse heart), carbonic anhydrase I (human erythrocytes) and P-lactoglobulin A (bovine milk). The effect of a moving ionic boundary inside the electrophoresis capillary on the separation resolution of model proteins by CZE/ESMS and CIEF/ESMS is described. In CZE/ESMS, the formation of a moving ionic boundary due to the replacement of background electrolyte counterions with sheath liquid counterions can be eliminated by using a common counterion in both the background electrolyte and the sheath liquid. Additionally, the moving ionic boundary in CIEF/ESMS is minimized by combining cathodic mobilization with a gravity-induced hydrodynamic flow. The concentration detection Limits of model proteins in a full-scan CIEF/ESMS analysis are in the region of M, N 20-50 times lower than that achievable using CZE/ESMS. Sample preconcentration taking place during the focusing step in CIEF is responsible for the improved detection limits.