Based on the synergy of sequential methotrexate (MTX) and 5-fluorouracil (5-FU) in vitro and in vivo and the superior antitumor activity of trimetrexate (TMTX) compared with MTX in preclinical models, we carried out a phase I trial of TMTX and 5-FU (fixed dose, 400 mg/m2 per day), both given as 10-min i.v. infusions daily x 5 days, every 28 days. The TMTX dose was escalated from 3.0 to 14.0 mg/m2 per day. In all, 92 evaluable courses were given to 34 patients, half of whom were heavily pretreated with radiation or cytotoxics. Myelosuppression and mucositis were the dose-limiting toxicities but were not different in heavily or minimally pretreated patients; there were five episodes of moderate to severe mucositis. Rash, fatigue, and diarrhea were mild toxicities. Plasma TMTX elimination was biexponential, with a mean t.1/2 alpha of 0.23 h and a t.1/2 beta of 16.7 h. The area under the plasma TMTX concentration versus time curve increased linearly with dose, suggesting first-order elimination. Total plasma TMTX clearance (mean +/- SD) was 14.3 +/- 5.9 ml/min per m2. Renal clearance accounted for approximately 7% of total clearance, indicating biotransformation as the major route of elimination. TMTX was highly protein-bound (97%). Thus, TMTX can be given with 5-FU (400 mg/m2) on a daily x 5-day bolus schedule at the 12 mg/m2 per day dose level, which was the recommended dose of TMTX as a single agent for phase II studies using the 5-day bolus schedule.