A threonine deaminase susceptible to inhibition by isoleucine was purified over 3,000-fold from extracts of Pseudomonas multivorans, a bacterium able to use threonine or α-ketobutyrate as sole source of carbon and energy. The enzyme was characterized with respect to molecular weight, dissociation to subunits, and apparent affinities for threonine, isoleucine, and several other ligands. Certain features of the enzyme including its reversible dissociation to subunits, its high constitutive activity, its marked stability, and high apparent orders of binding for threonine and isoleucine were unusual compared to those of isoleucine-inhibitable enzymes from other bacteria. These findings suggested some relationship between properties of the enzyme and the ability of P. multivorans to use threonine as sole carbon source. However, mutant studies ruled out a direct role of the enzyme in threonine catabolism and indicated that another enzyme, threonine dehydrogenase, is essential for growth on threonine.