Maple syrup urine disease in man and cattle is an inborn metabolic error caused by the deficiency of the branched chain alpha-ketoacid dehydrogenase. We have studied the molecular basis of the disease in Polled Hereford calves. The E1 component of branched chain alpha-ketoacid dehydrogenase was virtually undetectable by Western blot analysis of fibroblasts from an affected calf. Northern blot analysis failed to detect the E1 alpha mRNA species in the fibroblasts. Nevertheless, it was readily demonstrated by reverse transcription of RNA followed by polymerase chain reaction that the mRNA for the E1 alpha subunit was present in the cells, albeit at very low concentrations. Sequencing of the polymerase chain reaction-generated cDNA for the entire coding region of the E1 alpha subunit revealed a single base substitution at codon -6 (CAG to TAG). This mutation introduces a stop codon in the leader peptide of the E1 alpha subunit, resulting in the premature termination of translation. The mutation was verified by hybridization of the amplified cDNA fragments from two affected calves with allele-specific oligonucleotides. This finding explains the pathogenesis of maple syrup urine disease in this breed of cattle, which provides the only known animal model for the human disease. In addition, the results provide evidence for the effect of premature translation termination on reducing the steady-state mRNA level and the dependence of E1 beta protein stability on the co-expression of the E1 alpha.