A technique of two-dimensional polyacrylamide gel electrophoresis for the separation of plasma proteins was described in detail. Human plasma proteins were separated by isoelectric focusing followed by electrophoresis in a 4 to 21% or a 4 to 30% linear gradient slab gel. Urea or SDS was not used throughout the procedure, so that the analyses of proteins in their biologically active state were possible. By this technique, human plasma protein was resolved into more than 250 spots.