The hippocampus is an important brain region playing a central role in short-term memory formation. Information on protein expression in human hippocampus is limited and we therefore decided to generate a protein map of this pivotal brain structure thus providing protein building blocks and an analytical tool. Two-dimensional electrophoresis with in-gel digestion of protein spots followed by mass spectrometrical identification coupled to the Mascot search program was applied. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) or in special cases MALDI-TOF/TOF was used in order to warrant unambiguous identification. 266 protein spots representing 165 different proteins and 101 probable isoforms or post-translational modifications were identified. The identified proteins were categorized into several protein groups as metabolic, cytoskeleton, antioxidant, chaperone, signaling, transcriptional, proteolytic, synaptosomal and neuronal, hypothetical and miscellaneous structures. This human hippocampus protein map provides an analytical tool for hydrophilic and even relatively small-molecular-weight proteins and contributes to the knowledge on protein building blocks thus forming the basis for protein-chemical work in this brain area in health and disease.