W e have studied the molecular bases of maple syrup urine disease by analyzing the activity, subunit structure, mRNA sequence, and the genome of the affected enzyme. The branched chain alpha-keto acid dehydrogenase (BCKDH) activity in the patient was 4.2-4.5% of the control level. Immunoblot analysis revealed that the E2 subunit of BCKDH (Mr 52,000) was absent and another protein band with an Mr of 49,000 was present. We amplified the cDNA of the E2 subunit obtained from the patient's cell using the polymerase chain reaction method, then sequenced the amplified cDNA, in which a 78-bp deletion was identified. The consanguineous parents and a sister had two species of mRNA; the one corresponding to the normal E2 subunit and the other with a 78-bp deletion, whereas findings in a brother were normal. The molecular size of the translation products as deduced from the abnormal mRNA sequence was compatible with an abnormal protein band (Mr 49,000) detected in the patient's cells by immunoblot analysis. Analysis of genomic DNA of BCKDH-E2 subunit revealed that the 78-bp deletion in the mRNA was caused by an exon skipping due to a single base deletion in the 5'-splice donor site. As a result of the mutation, part of the inner E2 core domain was omitted. The specified region of the inner E2 core domain was highly homologous to the region of the E2 subunit of pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. These observations imply the biological importance of the region in the inner E2 core domain of BCKDH to maintain normal function of the activity.