Tyr 146 of TS has been proposed to assist in the removal of the proton from the 5-carbon of the pyrimidine in a steady-state intermediate [Hyatt, D. C., Maley, F., and Montfort, W. R. (1997) Biochemistry 36, 4585-4594]. We prepared a replacement set of mutations at position 146 of L. casei TS. The kcat and kcat/Km values of 15 mutants studied were significantly lower than wild-type TS. There was no effect on the Km of dUMP, and only moderate effects on the Km of the cofactor. We concluded that Y146 is not directly involved in substrate binding, but contributes significantly to catalysis. We also examined the Y146 mutants as catalysts for cofactor-independent dehalogenation of BrdUMP, a reaction which simulates early steps of the normal pathway up to and including enzyme-nucleotide covalent adduct formation. Many mutants had activity comparable to the wild-type enzyme, and we concluded that the effects of Tyr 146 mutations occur after the initial covalent adduct is formed. A covalent steady-state intermediate-containing enzyme, dUMP, and cofactor accumulated with Tyr 146 mutants, and could be isolated by SDS-PAGE. The complex was kinetically competent as an intermediate in dTMP formation. Using Y146D and F, it was shown that removal of the C-5 proton from the covalent intermediate was defective. We conclude that in the wild-type enzyme Tyr 146 assists in proton removal from the covalent intermediate. Mutants containing fluorinated tyrosines at position 146 showed an inverse linear correlation of activity versus acidity, again indicating that the basicity of the phenolic oxygen plays an important catalytic role. Speculations of how the poorly basic phenol group might assist proton removal are made in which Tyr 146 acts as a proton conduit to N5 of the cofactor or as a cohort of a water molecule serving as the direct general base catalyst.