Twenty clones stably resistant to 5-fluorouracil, 5-fluoro-2'-deoxyuridine, or 5-fluorouridine have been isolated from L1210 or P388 murine leukemia cells by a one-step mutation and selection procedure. The activities of enzymes of the pyrimidine salvage pathway relevant to the activation of these drugs have been determined in order to elucidate the mechanisms of resistance in these cells. Cell line resistant to 5-fluorouracil have 7 to 50% of the pyrimidine phosphoribosyltransferase activity found in the wild-type cells, with 5-fluorouracil, uracil, or orotate as substrate. Cells selected for resistance to 5-fluoro-2'-deoxyuridine have no detectable thymidine kinase activity. 5-Fluorouridine-resistant cells have 3 to 25% of the uridine kinase activity measured in the wild-type cell lines. No significant changes were observed in the activities of thymidylate synthetase, nucleoside phosphorylases, or 5-fluorouridylate kinase in any of the resistant cell lines. These findings have relevance to the treatment of human cancer, since pyrimidine phosphoribosyltransferase, thymidine kinase, or uridine kinase could be assayed in tumor biopsies in order to predict whether the fluoropyrimidines would be effective in individual patients.