We have identified amplification of the thymidylate synthase gene in a colonic tumor that had developed resistance to 5-fluorouracil/leucovorin combination chemotherapy. The tumor had previously undergone a partial response to this combination but began to progress following a prolonged period of continuous therapy and relative disease stabilization. Since thymidylate synthase is a target enzyme for 5-fluorouracil, it is likely that the observed gene amplification is responsible for the resistance. Thus, gene amplification may be a relevant mechanism of acquired resistance to fluoropyrimidine chemotherapy in the clinic.