Barley endo-beta-1,4-xylanases (EC 188.8.131.52) hydrolyse arabinoxylan, which is an abundant cell wall constituent in grass species. In addition to isoenzyme X-1, the major endo-beta-1,4-xylanase released from the aleurone layer of germinating barley, another isoenzyme, named X-II, is expressed during germination. Using PCR on barley genomic DNA, we isolated the complete gene encoding sequence of this less characterized isoenzyme. The gene, shown to be located on chromosome arm 5HL, has a coding sequence of 1665 bp interrupted by two introns. The corresponding protein sequence shows a high overall homology to X-I (88%) and reveals a modular structure consisting of a carbohydrate binding module and a glycosyl hydrolase family 10 domain but no signal peptide. Isoenzyme-specific sequence differences were exploited to develop X-I- and X-II-specific reverse transcriptase-PCR assays. These showed that X-I and X-II portray distinct expression profiles throughout plant development. Both isoenzymes are transcribed during germination but, in contrast to X-I, X-II transcripts accumulate in the developing shoot and root of the seedling embryo and not in the aleurone layer surrounding the endosperm. During subsequent stages of development, up to anthesis, X-II remains expressed in various organs. In developing grains, X-II is transcribed in the early stages of grain filling whereas X-I transcription is switched on during the later stages. The possible implications of these findings are discussed in regard to the distinct physiological role of the two isoenzymes. (c) 2005 Elsevier Ireland Ltd. All rights reserved.