Membrane proteins were separated by high resolution two-dimensional (2-D) electrophoresis. On isoelectric focusing (IEF) with immobilized pH gradients severe protein losses in the resulting 2-D map were observed when compared with carrier ampholyte-based IEF. This has been noticed for two different biological systems, namely the chloroplast envelope of spinach and the endocytic vesicles from Dictyostelium discoideum. The possible mechanisms of these losses on immobilized pH gradients are discussed.