The HOM3 gene of Saccharomyces cerevisiae encodes aspartate kinase, which catalyses the first step in the branched pathway leading to the synthesis of threonine and methionine from aspartate. Regulation of the carbon flow into this pathway takes place mainly by feedback inhibition of this enzyme by threonine. We have isolated and characterized three HOM3 mutants that show growth inhibition by threonine due to a severe, threonine-induced reduction of the carbon flow into the aspartate pathway, leading to methionine limitation. One of the mutants has an aspartate kinase which is 30-fold more strongly inhibited by threonine than the wild-type enzyme. The predicted amino acid substitution in this mutant, A406T, is located in a region associated with the modulation of the enzymatic activity. The other two mutants carry an aspartate kinase with reduced affinity for its substrates, aspartate and ATP. The corresponding amino acid substitutions, K26I and G25D, affect residues located in the vicinity of a highly conserved lysine-phenylalanine-glycine-glycine (KFGG) stretch present in the N-terminal part of the aspartate kinase, to which no function has so far been assigned. We suggest that this region is involved in substrate binding. Mutagenesis of a HOM3 region centred in the KFGG-coding triplets generated alleles that determine threonine sensitivity or auxotrophy for threonine and methionine, but not a phenotype associated with a feedback-resistant aspartate kinase, indicating that this region is not involved in the allosteric response of the enzyme.