The alpha-aminoadipate pathway of lysine biosynthesis is modulated at the transcriptional and biochemical levels by feedback inhibition. The first enzyme in the alpha-aminoadipate pathway, homocitrate synthase (HCS), is the target of the feedback regulation and is strongly inhibited by l-lysine. Here we report the structure of Schizosaccharomyces pombe HCS (SpHCS) in complex with l-lysine. The structure illustrates that the amino acid directly competes with the substrate 2-oxoglutarate for binding within the active site of HCS. Differential recognition of the substrate and inhibitor is achieved via a switch position within the (alpha/beta)(8) TIM barrel of the enzyme that can distinguish between the C5-carboxylate group of 2-oxoglutarate and the epsilon-ammonium group of l-lysine. In vitro and in vivo assays demonstrate that mutations of the switch residues, which interact with the l-lysine epsilon-ammonium group, abrogate feedback inhibition, as do substitutions of residues within the C-terminal domain that were identified in a previous study of l-lysine-insensitive HCS mutants in Saccharomyces cerevisiae. Together, these results yield new insights into the mechanism of feedback regulation of an enzyme central to lysine biosynthesis.