The kinetic mechanism of homoserine kinase, purified to homogeneity from Escherichia coli, was examined by initial velocity techniques at pH 7.6. Whereas ATP displayed normal Michaelis-Menten saturation kinetics ( K m = 0.2 mM), l-homoserine showed hyperbolic saturation kinetics only up to a concentration of 0.75 m m ( K m = 0.15 mM). Above this concentration, l-homoserine caused marked but partial inhibition ( K i ~ 2 mM). The kinetic data indicated that the addition of substrates to homoserine kinase occurs by a preferred order random mechanism, with ATP preferentially binding before l-homoserine. When the ATP concentration was varied at several fixed inhibitory concentrations of l-homoserine, the resulting inhibition pattern indicated hyperbolic mixed inhibition. This suggested a second binding site for l-homoserine. l-Aspartate semialdehyde, an amino acid analog of l-homoserine, proved to be an alternative substrate of homoserine kinase ( K m = 0.68 mM), and was subsequently used as a probe of its kinetic mechanism. In aqueous solution, at pH 7.5, this analog was found to exist predominantly (ca 85%) as its hydrated species. When examined as an inhibitor of the physiological reaction, l-aspartate semialdehyde showed mixed inhibition versus both l-homoserine and ATP. Although the pH profiles for the binding of l-homoserine as a substrate ( K m ) and as an inhibitor ( K i ) were identical, the kinetic data were best fit to a two-site model, with separate catalytic and inhibitory sites for l-homoserine.