Barley is a major cereal crop grown mainly for feed and malting. Two-dimensional gel electrophoresis has been used to analyse barley proteins for over 20 years and more recently, mass spectrometry was applied. In the absence of a genome sequence, barley gene and EST sequences combined with information from rice and other cereals facilitate identification of barley proteins. Several hundred barley seed proteins are identified and lower abundance proteins including membrane proteins are now being analysed. In the present review we focus on variation in protein profiles of seed tissues during grain filling, maturation, germination and radicle elongation. Cultivar comparisons and genetic mapping of polymorphic protein spots in doubled haploid populations provide a means to link the genome to the proteome and identify proteins that can influence grain quality. Many proteins appear in multiple forms on 2D-gels. Specific protein families, including peroxidases and alpha-amylases have been subjected to in-depth analysis resulting in characterisation of different isozymes, post-translational modifications and processing. A functional proteomics study focusing on the seed thioredoxin system has led to identification of thioredoxin target proteins, quantitative analysis of reduction of individual target disulphides and structural studies to gain insight into determinants for target protein recognition by thioredoxin.