A new form of microchip isoelectric focusing that allows efficient coupling with pretreatment processes is reported. The sample is conveyed in a carrier ampholyte solution to the separation channel that is connected at both ends by two V-shaped lead channels, which supply electrode solutions to the connection point and complete the electrical connection to off-chip electrodes. The relatively high electric conductivity of the electrode solutions compared with that of the pH gradient enables focusing with a 2% loss of applied voltage at the electrodes using the lead channels. A glass microchip was constructed specifically for this configuration. The channel wall was coated with polydimethylacrylamide, and the IEF chip was operated in a chip holder equipped with on-chip connector valves. A plug of fluorescence-labeled peptide p I markers with p I values ranging from 3.64 to 9.56 with carrier ampholyte solution (pH 3-10) was introduced into the separation channel. When the plug reached the channel segment (24 mm in length) between the connection points with the electrolyte lead channels, isoelectric focusing was started after filling the lead channels with electrolyte solutions. The peptide markers were observed using scanning fluorescence detection. The entire range of the pH gradient was established in the segment after approximately 2 min. Isoelectric focusing of three consecutively injected sample plugs containing different p I markers was demonstrated.