Saccharomyces cerevisiae aspartate kinase (AK(Sc)) phosphorylates L-Asp as the first step in the aspartate pathway responsible for the biosynthesis of L-Thr, L-Met, and L-Ile in microorganisms and plants. Using site-directed mutagenesis, we have evaluated the importance of residues in AK(Sc) that are strongly conserved among aspartate kinases or in other small molecule kinases. Steady state kinetic analysis of the purified AK(Sc) variants reveals that several of the targeted amino acids, particularly K18 and H292, have important roles in the enzymatic reaction. These results provide the first identification of amino acid residues crucial to the action of this important metabolic enzyme.