We have determined the kinetic parameters of human recombinant thymidylate synthase (hrTS) with its natural substrate, dUMP, and E-5-(2-bromovinyl)-2'-deoxyuridine monophosphate (BVdUMP), a nucleotide derivative believed to be the active species of the novel anticancer drug NB1011. NB1011 is activated by hrTS and is selectively toxic to high thymidylate synthase expressing tumor cells. BVdUMP undergoes hrTS-catalyzed thiol-dependent transformation. dUMP and BVdUMP act as competitive hrTS substrates. The natural folate cofactor, CH2-THF, inhibits the TS-catalyzed reaction with BVdUMP. We suggest that lower folate levels found in tumor cells favor TS-catalyzed BVdUMP transformation, which, in addition to higher levels of TS expression in tumor cells, contributes to the favorable therapeutic index of the drug NB1011. (C) 2003 Elsevier Inc. All rights reserved.