The human milk fat globule membrane protein composition is still largely unknown, although it counts for 2-4% of the total milk protein content and contains several important biologically active components. The aim of this work was to create a two-dimensional electrophoresis (2-DE) map of the human milk fat globule membrane proteins, both integral and membrane-associated, and to identify and characterize as many protein components as possible. A new protocol for the solubilization and extraction of the human milk fat globule membrane proteins with a double extraction procedure is presented, and the results compared with the extraction methods reported in the literature. The proteins were separated, in the first dimension, by isoelectric focusing (IEF) in the pH range 3-10 on strips of 13 cm length and, in the second dimension, by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 11.5% T homogeneous gels. A reproducible 2-DE map of integral and membrane-associated proteins was obtained and the first 23 spots, representing the major components, were identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometric analysis and/or by amino acid sequencing.