Germination of monocotyledonous plants involves activation and de novo synthesis of enzymes that degrade cell walls and starch and mobilize stored endosperm reserves for embryo growth. Two-dimensional (2-D) gel electrophoresis and mass spectrometry were applied to identify major water-soluble proteins in extracts of mature barley (Hordeum vulgare) seeds and to follow their fate during germination. About 1200 and 600 spots of p/ 4-7 were detected on 2-D gels by silver staining and colloidal Coomassie Brilliant Blue staining, respectively. About 300 spots were selected for in-gel digestion followed by matrix-assisted laser desorption/ionization-mass spectrometry-peptide map fingerprint analysis. Database searches using measured peptide masses resulted in 198 identifications of 103 proteins in 177 spots. These include housekeeping enzymes, chaperones, defence proteins (including enzyme inhibitors), and proteins related to desiccation and oxidative stress. Sixty-four of the identifications were made using expressed sequence tags (ESTs). Numerous spots in the 2-D gel pattern changed during germination (micromalting) and an intensely stained area which contained large amounts of the serpin protein Z appeared centrally on the 2-D gel. Spots containing a-amylase also appeared. Identification of 22 spots after three days of germination represented 13 different database entries and 11 functions including hydrolytic enzymes, chaperones, housekeeping enzymes and inhibitors.