In order to characterize a -amylase deficiency in the endosperm of mutant rye lines, homologous cDNA probes were prepared. A rye cDNA library was constructed from a normal line and screened with a barley -amylase probe. Three partial cDNA clones specific for endosperm -amylase in rye were isolated and characterized. The largest of these clones was used to investigate the expression of endosperm -amylase in mutant and normal lines by Northern hybridization. These experiments, as well as in vitro translation experiments, demonstrate the absence of endosperm -amylase mRNA in mutant lines. Sequencing of three different cDNA clones revealed a single nucleotide difference, which suggests that two genes encoding endosperm -amylase genes might exist in rye. From Southern blots we anticipate that these two genes are tightly linked. Results of these experiments and previous data indicating that the mutation was located within the -amylase locus on chromosome 5 are consistent with the hypothesis that the mutation results from a deletion simultaneously affecting the two genes. However, due to extensive polymorphism within normal lines used as control, additional experiments will be required to further substantiate this conclusion. The deduced amino acid sequence reveals the occurrence of three short glycine-rich repeats containing 11 or 12 residues close to the carboxyl terminus of the protein. A comparison of the nucleotide sequence between rye and previously described barley cDNA clones revealed ca. 90% homology at the amino acid level, except in this C-terminal repeated part, where it drops to 45%.