X-ray crystal structures of binary complexes of dUMP or dCMP with the Lactobacillus casei TS mutant N229D, a dCMP methylase, revealed that there is a steric clash between the 4-NH2 of dCMP and His 199, a residue which normally H-bonds to the 4-O of dUMP but is not essential for activity. As a result, the cytosine moiety of dCMP is displaced from the active site and the catalytic thiol is moved from the C6 of the substrate about 0.5 Å further than in the wild-type TS−dUMP complex. We reasoned that combining the N229D mutation with mutations at residue 199 which did not impinge on the 4-NH2 of dCMP should correct the displacements and further favor methylation of dCMP. We therefore prepared several TS N229D mutants and characterized their steady state kinetic parameters. TS H199A/N229D showed a 1011 change in specificity for methylation of dCMP versus dUMP. The structures of TS H199A/N229D in complex with dCMP and dUMP confirmed that the position and orientation of bound dCMP closely approaches that of dUMP in wild-type TS, whereas dUMP was displaced from the optimal catalytic binding site.