PALA is an inhibitor of de novo pyrimidine biosynthesis. Studies combining PALA with 5-FU in experimental models have demonstrated synergistic antitumor activity with only additive toxicity. The phase I study of PALA in combination with 5-FU is described. Sixteen patients received a total of 29 courses of PALA given as a 24-hour infusion daily for 5 days and 5-FU given by iv bolus at the end of each 24-hour PALA infusion. Cycles were repeated at 28-day intervals. Mucositis was dose-limiting when 940 mg/m3/day of PALA was given with 345 mg/m2/day of 5-FU. Diarrhea, skin rash, and myelosuppression (in decreasing order of frequency) occurred but were not dose-limiting. Alopecia occurred in all patients. Objective responses were seen in single patients with large cell carcinoma of the lung, fibrous histiocytoma, and adenocarcinoma of the colon refractory to prior 5-FU therapy. These studies support further phase II evaluation of the PALA and 5-FU combination.