A library of double stranded cDNA clones was constructed using mRNA isolated from developing barley endosperms as template. The cDNA clones were classified by restriction endonuclease mapping and by hybridization to single stranded cDNA prepared from mRNA of two hordein deficient mutants of barley. This classification and nucleotide sequence analysis identified cDNAs coding for B1 hordein polypeptides. Hybridization of selected cDNA sequences to a B1 hordein cDNA probe at different conditions of stringency demonstrated the presence of a group of related but not identical sequences. Hybridization of the B1 hordein cDNA probe to restriction endonuclease fragments of barley nuclear DNA suggests that the B1 hordein polypeptides are encoded by a multigene family. Abundant mRNA sequences ranging in size from 1,200 to 1,400 nucleotides were detected by hybridization to the B1 hordein cDNA probe. This size is sufficient to code for the B1 hordein polypeptide precursor, which is estimated to have a molecular weight in the range of 29,000 to 35,000. Mutant Risø 56 (hor2ca), which is defective in the synthesis of B hordein polypeptides, lacked B1 hordein mRNAs, thus strongly indicating that the mutation prevents the transcription of these genes. Mutant Risø 1508 (lys3a), which is deficient in the synthesis of all hordein polypeptides, contained detectable amounts of RNA sequences homologous to the B1 hordein cDNA, although the hybridization level was reduced in comparison to the wild type. Failure of these RNA sequences to be translated into B1 hordein polypeptides in a cell free protein synthesizing system hints that the lys3a gene is involved in the post-transcriptional modification of the messenger RNA. Major deletions were not detected in the gene cluster coding for B hordein polypeptides by hybridization of the B1 hordein cDNA to restriction endonuclease fragments of the nuclear DNA from the two mutants Risø 56 and Risø 1508.