The cytotoxic effects of the antifolates methotrexate (MTX) and trimetrexate (TMQ) were investigated for two human leukemic CCRF-CEM cell lines, one expressing the "classical" reduced folate/MTX carrier (CEM-RF), another lacking this carrier but expressing a membrane associated folate binding protein (CEM-FBP). CEM-FBP cells were found to be highly resistant to MTX compared to CEM-RF cells, especially in short exposures. For example, after 4 h incubation, IC50 values for MTX were 251 microM and 0.98 microM for CEM-FBP and CEM-RF cells, respectively. On the other hand, CEM-FBP cells were much more sensitive to the lipophilic antifolate TMQ than CEM-RF cells as shown by IC50 values (after 4 h of exposure) of 0.059 microM and 7.5 microM, respectively. Finally, the reversal of TMQ cytotoxicity by folinic acid was significantly impaired for CEM-FBP cells, in contrast to CEM-RF cells. These results indicate that the nature of the membrane transport system for folates can be a critical determinant in tumor cell sensitivity or resistance to antifolates.