Thymidylate (dTMP) synthetase (EC 2.1.1.45) catalyzes the conversion of 2'-deoxyuridylate (dUMP) and 5,10-methylenetetrahydrofolate (CH2-H4folate) to 2'-deoxythymidylate (dTMP) and 7,8-dihydrofolate. This enzyme represents the sole de novo pathway for dTMP synthesis and has received much attention as a target for inhibitors with potential chemotherapeutic value. The catalytic mechanism and inhibition of this enzyme have recently been reviewed. One class of potent inhibitors of this enzyme is 5-substituted dUMP's, which act as mechanism-based inhibitors. An early event in the normal enzymatic reaction involves nucleophilic attack of a cysteine thiol of the enzyme at the 6 position of dUMP to form 5,6-dihydropyrimidine intermediates which are covalently bound to the enzyme during the remaining catalytic sequence. The 5-substituted dUMP's, which are mechanism-based inhibitors of this enzyme, undergo similar nucleophilic attack at the 6 position; subsequently, the analogue either remains attached to the enzyme or a moiety at the 5 position of the inhibitor is activated so that it may covalently interact with the enzyme. α,β-Acetylenic carbonyl compounds have received much attention as suicide inactivators of enzymes. The acetylenic functional group is normally inert toward nucleophiles, but enzyme-catalyzed generation of a carbanion at the α carbon can result in isomerization to a conjugated allene; the latter is a powerful Michael acceptor, and if a nucleophile of the enzyme is juxtaposed to the reactive β carbon, covalent bond formation can occur. As suggested by other workers, since the initial covalent bond formation between dTMP synthetase and 5-substituted dUMP's generates a transient carbanion at the 5 position, 5-alkynyl-dUMP's are potential suicide inactivators of this enzyme. 5-Ethynyl-dUrd (EdUrd) has recently been prepared and shown to be a potent growth inhibitor of cells grown in tissue culture; in addition, a preparation of 5-ethynyl-dUMP (EdUMP) was found to inhibit dTMP synthetase. Unfortunately, the tedious synthesis of these compounds precluded a thorough evaluation of EdUMP, and its mechanism of action was unresolved. Recently, a facile, general synthesis of 5-alkynyl-dUrd's has been reported. Here, we describe preliminary observations of the inhibitory properties of a number of 5-alkynyl-dUMP's toward dTMP synthetase, as well as the growth inhibitory properties of the corresponding nucleosides toward S-49 mouse lymphoma cells.