Because leucovorin increases the antitumor activity of 5-fluorouracil (5-FU) in multiple tumor model systems, we performed a clinical trial to evaluate this combination in women who had received one or no prior chemotherapy regimens for metastatic breast cancer. Thirty-six women with measurable metastatic disease were treated with five consecutive days of i.v. leucovorin, 500 mg/m2/day infused over 30 min, followed 1 h later by i.v. bolus 5-FU, 375 mg/m2/day. Repeat cycles were planned at 4-week intervals. Tumor regression occurred in 10 of 36 patients (28%; 95% confidence interval, 14-45%) with a median time to disease progression (TTP) of 8.7 months (range 3.2-16.8 months) in the responding patients. The median TTP and survival for all patients were 3.0 and 12.4 months, respectively. Among 30 patients who had received prior 5-FU, tumor regressions were seen in seven (23%; 95% confidence interval 10-42%). The major dose-limiting toxicity in this study was mucositis, affecting 89% of the patients. Other toxicities were tolerable in the majority of patients. Although the role of leucovorin in breast cancer clinical practice remains undefined, the data from this trial support the hypothesis that leucovorin enhances the cytotoxic activity of 5-FU against human breast cancer.