Proteomes of barley seeds were described by 2-D gel electrophoresis and spots selected for proteinidentification by mass spectrometry and database searches. Proteins were categorised according to temporalappearance during seed development and maturation. Fragments of β-amylases appeared transiently at midgrain filling and during germination. The α-amylase/trypsin inhibitors increased during grain filling andtypical housekeeping enzymes were present throughout the period. Germination altered the proteome and dissection of micromalted seeds enabled localization of selected proteins to specific tissues. The embryo was particularly rich in soluble proteins. Barley α-amylase/subtilisin inhibitor (BASI) and its target α-amylaseisozyme 2 occurred in endosperm and aleurone. Immunoblotting showed how the α-amylase changed duringgermination. Mutational analysis in a-amylase and BASI was guided by the three-dimensional structures.Mutants along the 10 binding subsites in barley a-amylase 1 addressed roles of individual residues in thepreference for starch over oligosaccharide substrates and vice versa, action patterns on oligosaccharides, andmultiple attack on amylose. Although the wild-type enzyme was proficient in transglycosylation, mutants ofthe catalytic nucleophile did not act as glycosynthase. C-terminal fusion of a starch binding domain from Aspergillus niger glucoamylase to barley α-amylase isozyme 1 enhanced degradation of starch granules. Recent heterologous expression of BASI allowed mutation of residues critical in enzyme binding as monitored by activity inhibition and surface plasmon resonance assays. A fully hydrated Ca²⁺ at the protein interface secured contact between BASI and the catalytic residues in the α-amylase.