Heteroxylans are major constituents of cell walls in the graminaceous monocotyledons. Degradation of walls in the starchy endosperm of germinated cereal grains is mediated, in part at least, by the action of (1-->4)-beta-xylan endohydrolases (EC 126.96.36.199). Complementary DNAs encoding (1-->4)-beta-xylan endohydrolases from the aleurone layer of germinated barley have been isolated and characterized. Southern blot analyses suggest that the enzymes are derived from a family of 3 or 4 genes, and cDNAs corresponding to two of these genes have been sequenced. The amino acid sequence deduced from one cDNA almost exactly matches the amino acid sequence determined previously from the purified enzyme. This enzyme is designated (1-->4)-beta-xylan endohydrolase isoenzyme X-I. The mature enzyme consists of 395 amino acid residues, has a calculated M(r) of ca. 44600 and an isoelectric point of 6.1, and is likely to adopt an (alpha/beta)8 barrel conformation. The amino acid sequence of the barley (1-->4)-beta-xylan endohydrolase encoded by the other cDNA, which is designated isoenzyme X-II, shows ca. 13% sequence divergence compared with isoenzyme X-I. Both enzymes exhibit sequence and structural similarities with microbial xylanases. Expression of the genes in germinated grain appears to be confined largely to the aleurone layer, and no mRNA transcripts could be detected in young vegetative tissues.