We have characterized a novel mutation in a male patient that affects the coding sequence of PDH-E₁α gene and changes arginine-141 to a leucine. This nucleotide substitution was found in about 75% of the studied DNA (fibroblasts, liver and muscle), a scenario that would indicate a case of E₁α mosaicism in a male patient. When the mutant E₁α protein was expressed in human skin fibroblasts with zero endogenous pyruvate dehydrogenase complex activity and E₁α protein expression, no significant restoration of activity was recorded, in contrast to the wild-type cDNA, even though both wild-type and mutant protein levels were comparable. We concluded that the R141L mutation is a severe one and that it must have occurred in one of the E₁α alleles during early embryogenesis.