Four hr infusions i.v. of [6RS]5-formyltetrahydrofolate ([6RS]5-CHO-H4PteGlu; 500 mg/m2) and [6RS]5-methyltetrahydrofolate ([6RS]5-CH3-H4PteGlu; 500 mg/m2) were compared for their relative effects on expansion of pools of 5,10-methylenetetrahydrofolates (CH2-H4PteGlun) and tetrahydrofolates (H4PteGlun) in two human colon adenocarcinoma xenografts in mice. Expansion of these pools by 253-661% of control and increase in predominance of di-, tri-, and tetra-glutamate species were observed during [6RS]5-CHO-H4PteGlu infusion. In contrast, only modest pool size expansion (148-164% of control) and limited modulation of polyglutamate species were detected in four tumor lines during infusion with [6RS]5-CH3-H4PteGlu. The data suggest that [6RS]5-CH3-H4PteGlu is less effective than [6RS]5-CHO-H4PteGlu as a precursor for pools of CH2-H4PteGlun and H4PteGlun in colon tumors.