Aspergillus nidulans is able to use Leu as the sole carbon source through a metabolic pathway leading to acetyl-CoA and acetoacetate that is homologous to that used by humans. mccA and mccB, the genes encoding the subunits of 3-methylcrotonyl-CoA carboxylase, are clustered with ivdA encoding isovaleryl-CoA dehydrogenase, a third gene of the Leu catabolic pathway, on the left arm of chromosome III. Their transcription is induced by Leu and other hydrophobic amino acids and repressed by glucose. Phenotypically indistinguishable DeltamccA, DeltamccB, and DeltamccA DeltamccB mutations prevent growth on Leu but not on lactose or other amino acids, formally demonstrating in vivo the specific involvement of 3-methylcrotonyl-CoA carboxylase in Leu catabolism. Growth of mcc mutants on lactose plus Leu is impaired, indicating that Leu metabolite(s) accumulation resulting from the metabolic block is toxic. Human patients carrying loss-of-function mutations in the genes encoding the subunits of 3-methylcrotonyl-CoA carboxylase suffer from methylcrotonylglycinuria. Gas chromatography/mass spectrometry analysis of culture supernatants revealed that fungal Deltamcc strains accumulate 3-hydroxyisovaleric acid, one of the diagnostic compounds in the urine of these patients, illustrating the remarkably similar consequences of equivalent genetic errors of metabolism in fungi and humans. We use our fungal model(s) for methylcrotonylglycinuria to show accumulation of 3-hydroxyisovalerate on transfer of 3-methylcrotonyl-CoA carboxylase-deficient strains to the isoprenoid precursors acetate, 3-hydroxy-3-methylglutarate, or mevalonate. This represents the first reported genetic evidence for the existence of a metabolic link involving 3-methylcrotonyl-CoA carboxylase between isoprenoid biosynthesis and Leu catabolism, providing additional support to the mevalonate shunt proposed previously (Edmond, J., and Popják, G. (1974) J. Biol. Chem. 249, 66-71).