Recent demonstrations that deazafolate analogues may act as potent inhibitors of thymidylate synthase (TS) provided a firm rationale for the synthesis of N10-propargyl derivatives of 8-deazafolate and 8-deazaaminopterin (4). A complete assignment of the 1H NMR spectra of these compounds was made possible through application of 2D (COSY) techniques at 200 MHz. Data describing the inhibition of TS derived from human leukemia (K562) cells are presented. IC50 values of 2.25 and 1.26 microM were determined for 8-deaza-10-propargylfolate (3) and 8-deaza-10-propargylaminopterin, respectively. Comparison of the data for various folate analogues reveals a striking dependence of TS inhibitory potency upon the number of nitrogens in the folate pyrazine ring.