Homoserine kinase [EC 2.7.1.39] was partially purified about 40-Iold from sonic extracts of Brevibacterium flavum. A new assay method for homoserine kinase was examined in which ADP formed in the reaction was measured in a coupled spectrophotometric assay with pyruvate kinase [EC 2.7. 1.40] and with lactate dehydrogenase [EC 1.1. 1.27]. The optimum pH of the reaction was 7.4 with Tris buffer, and the Km values at pH 7.5 for the substrates, homoserine and ATP, were 7.7×10−4M and 1.2×10−3M, respectively. Ammonium sulfate or KCl activated the enzyme about twice. Phosphohomoserine, one of the reaction products, inhibited the forward reaction competitively to both of the substrates, suggesting that the reaction mechanism was rapid equilibrium random Bi Bi, that is, random addition of two substrates and random release of two products with rapid equilibrium. Threonine, a metabolic end product, inhibited the activity competitively to homoserine and not competitively to ATP, and 50% inhibition took place at about 8mM of threonine. A gel filtration experiment showed that the enzyme was neither aggregated with aspartate kinase nor with homoserine dehydrogenase and its molecular weight was about 5.5×104. The properties of homoserine kinase of a threonine-analogue-resistant-mutant from B. flavum, which overproduces threonine in the culture medium, were essentially the same as those of the parental enzyme.