Six peptides were isolated from glycogen debranching enzyme purified from rabbit muscle, and their sequences were determined. A cDNA library made from rabbit muscle using random hexamer primers was screened with oligonucleotide probes constructed in accordance with these peptide sequences. Seven cDNA clones comprising the open reading frame were found, whereas oligo(dT) cDNA libraries yielded no positive clones because of the long 3'-nontranslated region of 2.3 kb. The open reading frame of 4665 bases codes for a 1555-amino-acid protein of M(r) 177,542. Compared to the sequence from human muscle, there are an additional 40 amino acid residues upstream from the N-terminus, and the next 10 residues show no homology. For the remaining 1505 residues, the two sequences exhibit an identity of 93%. The four consensus sequences commonly found at the carboxy termini of beta-strands in the alpha/beta barrel domains of amylases and glucanotransferases are also found in the N-terminal half of the debranching enzyme, suggesting that this structural domain may be present. This and other evidence suggests that the N-terminal half may encompass the transferase activity, leaving the glucosidase activity for the C-terminal half. The latter shows no significant homology to known proteins. An unusual feature of the sequence is the presence of three pairs of adjacent cysteines, which may explain inhibition of the enzyme by organic arsenites.