Twenty-one patients affected by advanced carcinoma of the digestive tract, all but 2 previously treated, received on day 1 every 2 weeks a 2-hour intravenous (i.v.) infusion of methotrexate (MTX), 250 mg/m2, followed 24 h later by a 2-hour i.v. infusion of L-folinic acid (LFA), 250 mg/m2, and 5-fluorouracil (FU), 600 mg/mg2 as an i.v. bolus. Only 1 previously untreated patient obtained a partial response. The MTX serum level assessed 24 h after its infusion (24-hour sMTX) ranged from 0.3 to 5.7 (median: 0.9) microM, and in only 8/21 patients reached a concentration > or = 1 microM. A further 46 patients (of whom 22 had been previously treated) received the same treatment as above but with a double dosage (500 mg/m2) of MTX. Twelve of these 46 patients (26%, 95% confidence interval = 14-41%) achieved a partial response with this regimen. Responses were obtained in chemotherapy-naive patients (8/24) and in previously treated patients (4/22). The 24-hour sMTX ranged from 1.2 to 9.5 microM)(median: 2.3) and was > or = 2 microM in 30/46 patients. Among patients showing a 24-hour sMTX value > or = 2 microM, the response rate was 39% (45% in previously untreated patients), while no patient with a 24-hour sMTX value below 2 microM at 24 h obtained a major response (p = 0.0017). Our findings demonstrate that 500 mg/m2 of MTX given as a 2-hour i.v. infusion is required to reach a serum concentration of at least 1 microM for 24 h. Furthermore, the double biochemical modulation of FU may obtain an objective response in patients previously treated with fluoropyrimidines.