Three inborn errors have been identified in the pathway of isoleucine degradation. Deficiency of beta-ketothiolase (beta-KT, also known as T2, mitochondrial acetoacetyl-CoA thiolase and acetyl-CoA acetyltransferase 1) is a well-described disorder which presents with acute episodic ketoacidosis. In contrast, short/branched-chain acyl-CoA dehydrogenase (SBCAD) and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiencies are recently described and relatively rare defects which present with predominantly neurological manifestations, although acute metabolic decompensation may occur in the early newborn period. Careful examination of urine organic acids is required for identification and differential diagnosis of these disorders, with awareness that the abnormalities may be subtle and variable. Tandem MS analysis of acylcarnitines may reveal elevated C5 (SBCAD) or C5:1 and/or OH-C5 species (MHBD and beta-KT deficiencies) but the abnormalities are non-diagnostic and may be intermittent or absent. Confirmation of diagnosis is therefore advisable by specific enzyme assay and/or mutation analysis of the ACAT1 (beta-KT), ACADSB (SBCAD) or HADH2 (MHBD) genes. The latter is located on the X chromosome, accounting for the milder clinical phenotype in females. If beta-KT deficiency is diagnosed early and treated by fasting avoidance and modest protein restriction, ketoacidosis episodes can be prevented and the prognosis is excellent. The role of treatment in SBCAD deficiency remains unclear pending further delineation of its clinical phenotype and pathogenicity, particularly regarding asymptomatic individuals detected by expanded newborn screening. The ineffectiveness of isoleucine restriction in MHBD deficiency is consistent with the additional roles of this multifunctional enzyme in sex steroid and neurosteroid metabolism and its interaction with amyloid-beta peptide.