A highly purified preparation of biosynthetic threonine dehydratase (l-threonine hydro-lyase (deaminating), EC 184.108.40.206) from Rhodopseudomonas spheroides, has been used for a kinetic study of its reaction mechanism in the absence and presence of modifiers. At pH 7.4 and in the absence of modifiers, the initial velocity data gave curvilinear double reciprocal plots and could be fitted to a rate equation that describes the addition of threonine to the enzyme at two interacting sites which catalyse substrate hydrolysis at different rates. The initial velocity data obtained in the presence of lower, but not higher, concentrations of isoleucine fitted well to the same equation. The inhibition of the reaction increased as a sigmoidal function of the isoleucine concentration and with threonine at a concentration of 40 mM, the data were consistent with a rate equation which is the ratio of second order polynomials. Enzyme activity increased as a hyperbolic function of the concentration of valine which was also capable of causing curvilinear double reciprocal plots to become linear. The latter effect was also observed with allothreonine that can act as a linear competitive inhibitor. Neither valine nor isoleucine affected the maximum velocity of the reaction and the inhibition by isoleucine could be reversed completely by valine. Attention has also been drawn to some of the inherent difficulties associated with the analysis of kinetic data for allosteric enzymes.