A procedure was established for micro-two-dimensional gel electrophoresis with agarose gels in the first dimension. Agarose (Agarose IEF, Pharmacia) was previously found to be suitable for the analysis of high molecular weight proteins with a wide range of isoelectric points, but rather difficult to be handled for micro-scale electrophoresis. However, addition of 1M thiourea in combination with 5M urea to the medium for agarose gel made it possible to be used with ease on micro-scale as well as on macro-scale. Since thiourea is a potent protein solubilizing reagent, almost all amounts of proteins including structural proteins were presented on the respective positions on two-dimensional gels. This method is applicable to other experiments with high molecular weight proteins to be analyzed in a wide range of isoelectric points.