Isovaleric acidemia (IVA) is an inborn error of leucine metabolism and is caused by a genetically determined deficiency of isovaleryl-CoA dehydrogenase (IVD), a mitochondrial matrix enzyme. IVD is produced as a 45-kDa precursor and then is transported into the mitochondria, where it is processed to its mature 43-kDa size. Previous [35S]methionine-labeling studies of fibroblasts from IVA patients have revealed at least five classes of mutations within the IVD gene. In size, IVD precursor and mature proteins produced by class I mutants are indistinguishable from their normal counterparts. Class II, III, and IV mutants make IVD precursor proteins which are 23 kDa smaller than normal. Subsequent processing in class III and IV mutants is normal but proceeds inefficiently in class II mutants. Class V mutants make no detectable IVD protein. In order to further study these mutations at the molecular level, the IVD coding region from mutant fibroblast cDNA was amplified by the PCR and was analyzed by DNA sequencing. cDNA from class I mutant alleles from two of seven class I mutant cell lines each contained a different missense mutation. In cDNA from a class III mutant, a single base deletion at position 1179 of the coding region was identified which leads to a shift in reading frame, predicting the incorporation of eight abnormal amino acids followed by a premature termination codon. Sequencing of amplified IVD cDNA from a type V mutant has failed to identify any abnormalities. It most probably is deficient in translation of the IVD mRNA. A new class of IVD mutant allele which appears to be transcriptionally defective (type VI) was also identified. Additional study of this set of IVD mutations should add both to our knowledge of the biosynthetic pathway of mitochondrial proteins and to our understanding of the clinical heterogeneity seen in IVA.