The metabolism of leucine to isoamyl alcohol in yeast was examined by 13C nuclear magnetic resonance spectroscopy. The product of leucine transamination, alpha-ketoisocaproate had four potential routes to isoamyl alcohol. The first, via branched-chain alpha-keto acid dehydrogenase to isovaleryl-CoA with subsequent conversion to isovalerate by acyl-CoA hydrolase operates in wild-type cells where isovalerate appears to be an end product. This pathway is not required for the synthesis of isoamyl alcohol because abolition of branched-chain alpha-keto acid dehydrogenase activity in an lpd1 disruption mutant did not prevent the formation of isoamyl alcohol. A second possible route was via pyruvate decarboxylase; however, elimination of pyruvate decarboxylase activity in a pdc1 pdc5 pdc6 triple mutant did not decrease the levels of isoamyl alcohol produced. A third route utilizes alpha-ketoisocaproate reductase (a novel activity in Saccharomyces cerevisiae) but with no role in the formation of isoamyl alcohol from alpha-hydroxyisocaproate because cell homogenates could not convert alpha-hydroxyisocaproate to isoamyl alcohol. The final possibility was that a pyruvate decarboxylase-like enzyme encoded by YDL080c appears to be the major route of decarboxylation of alpha-ketoisocaproate to isoamyl alcohol although disruption of this gene reveals that at least one other unidentified decarboxylase can substitute to a minor extent.