The interaction of dTMP synthetase with N4-hydroxy-2'-deoxycytidylate (N4-HOdCMP) has been investigated. With use of standard assay conditions, N4-HOd-CMP is a competitive inhibitor with an apparent Ki of 8.0 microM. Incubation of N4-HOdCMP with dTMP synthetase in the presence of 5,10-methylenetetrahydrofolate (CH2-H4folate) resulted in a rapid time-dependent inactivation of the enzyme which was not first order and the formation of complexes which could be isolated on nitrocellulose filter membranes. With use of radioactive ligands, the isolable native complex was shown to possess 2 mol of N4-HOdCMP and 2 mol of CH2-H4folate/mol of dimeric enzyme; the apparent dissociation constant of N4-HOdCMP was 1.0 microM. Ultraviolet difference spectroscopy of the ternary complex showed a loss of the pyrimidine chromophore which did not reappear upon denaturation with NaDodSO4. The rate of dissociation of N4-HOdCMP from the ternary complex was biphasic in which one-half of the initially bound ligand dissociated with t 1/2 congruent to 2.3 min and the remainder with t 1/2 congruent to 13 min. When the N4-HOdCMP-CH2-H4folate-enzyme complex was denatured, one-half of the CH2-H4folate dissociated whereas all of the N4-HOdCMP remained bound to the enzyme. Taken together, our results indicate that N4-HOdCMP forms a covalent bond with dTMP synthetase and reveal an unusual asymmetry in the two subunits of the N4-HOdCMP-CH2-H4folate-enzyme complex. It appears that one subunit is covalently bound to N4-HOdCMP, which, in turn, is covalently linked to CH2-H4folate whereas the other subunit is covalently bound to N4-HOdCMP but CH2-H4folate is bound by noncovalent interactions.