The first step in the biosynthesis of leucine is catalyzed by alpha-isopropylmalate (alpha-IPM) synthase. In the yeast Saccharomyces cerevisiae, LEU4 encodes the isozyme responsible for the majority of alpha-IPM synthase activity. Yeast strains that bear disruption alleles of LEU4, however, are Leu+ and exhibit a level of synthase activity that is 20% of the wild type. To identify the gene or genes that encode this remaining activity, a leu4 disruption strain was mutagenized. The mutations identified define three new complementation groups, designated leu6, leu7 and leu8. Each of these new mutations effect leucine auxotrophy only if a leu4 mutation is present and each results in loss of alpha-IPM synthase activity. Further analysis suggests that LEU7 and LEU8 are candidates for the gene or genes that encode an alpha-IPM synthase activity. The results demonstrate that multiple components determine the residual alpha-IPM synthase activity in leu4 gene disruption strains of S. cerevisiae.