- Leucovorin requirements for protection of the T cell line CCRF-CEM and the B cell line LAZ-007 against the cytotoxic effects of a variety of antifolates were studied. Differential leucovorin protection for DDMP-induced growth suppression occurred in the opposite direction to that for MTX, with CCRF-CEM requiring less leucovorin than LAZ-007 for equivalent protection. A pattern of differential protection from DDMP different from that of protection from MTX was also seen for the cell lines RAJI and MOLT-4. Differential leucovorin protection was observed for the chain-extended MTX analogue PT441. The degree of differential protection was similar to that seen for MTX, and transport studies showed that PT441 was a weak inhibitor of tritiated MTX uptake into CCRF-CEM cells. Differential leucovorin protection was observed for the lipophilic antifolate trimetrexate glucoronate (TMQ) but the degree of differential protection was smaller than that seen for PT441 or for MTX. Since TMQ is not transported into cells by the reduced folate system, while PT441 is a weak competitive inhibitor of [3H]MTX transport, and since neither is polyglutamylated, these results support the conclusion reached in previous experiments that differential leucovorin protection of MTX is unlikely to be a transport-related phenomenon and is not due to an effect on polyglutamylation. In addition, the different patterns of relative leucovorin requirements for DDMP and MTX protection suggest that differential metabolism or catabolism of leucovorin does not account for differential protection.