Thymidylate synthase has been isolated from MTX-resistant strains of Pseudomonas sp. KL-9, using a procedure that involves fractionation with ammonium sulfate, filtration through Sephadex G-150, and chromatography on CM-Sephadex. The specific activity of enzyme was $6.4{ imes}10^{-3}U/mg$ and the overall yield was about 15.3%. This enzyme showed a high substrate specificity on dUMP and THF, and required 2-mercaptoethanol and $Mg^{2+}$. In tris-acetate buffers, the enzyme displayed pH optimum at 6.5 when dUMP was the substrate. The optimal temperature was $40^{circ}C$ $K_{m}$ values for 5,10-methylenetetrahydrofolate and dUMP were $6.9{ imes}10^{-7}M$ and $6.6{ imes}10^{-8}M$, respectively, which were lower values than the case of L. casei TS.