We have examined the appearance of amplification of 2 genes involved in resistance to chemotherapy (thymidylate synthase, dihydrofolate reductase) in patients receiving endocrine treatment. Chronological tumor samples were obtained from breast-cancer patients with clinical stage-IV disease, using fine-needle biopsies. The presence of amplification of thymidylate synthase and dihydrofolate reductase was determined using PCR in 185 fine-needle biopsies from 37 patients. None of the initial samples of each patient showed amplification. However, in 7 of the 37 patients (19%) we detected development of gene amplification: in 2 cases of thymidylate synthase and in 5 cases of dihydrofolate reductase. Five of the 7 patients with amplification were receiving second-line endocrine treatment after failing to respond to tamoxifen.