Fluoroglutamate-containing analogs of folates and methotrexate (MTX) with altered capacities for poly (γ-glutamate) metabolism were synthesized to probe the biological roles of polyglutamates. Compared to folic acid, dl- e,t-γ-fluorofolic acid, a compound that is a poor substrate for polyglutamylation, was ≈25-fold less potent in promoting growth of folate-depleted H35 rat hepatoma cells. dl-β,β-Difluorofolic acid, a compound that forms diglutamates more readily than does folic acid, was at least equivalent to folic acid in potency. Leucovorin (LV), a reduced folate, was 30-fold more potent than folic acid in promoting growth, whereas the analogous form of dl- e,t-γ-fluorofolate, dl- e,t-γ-fluoroleucovorin ( dl- e,t-γ-FLV) was only 4-fold more potent than folic acid. Both LV and dl- e,t--γ-FLV protected or “rescued” cells from the growth inhibitory effects of MTX; however a 37- to 46-fold higher concentration of the fluoro analog was required. Folic acid, dl- e,t-γ-fluorofolic acid, LV, and dl- e,t-γ-FLV each potentiated the growth inhibitory effect of 5-fluoro-2′-deoxyuridine on CCRF-CEM human leukemia cells; higher concentrations of fluorinated analogs again were required. Stereochemically pure l- t-γ-fluoromethotrexate ( l- t-γ-FMTX), a poor substrate for polyglutamylation, was evaluated as a cell growth inhibitor. In continuous exposure, l- t-γ-FMTX was 7-fold less potent than MTX as an inhibitor of CCRF-CEM growth. Results with these fluorinated folate and MTX analogs offer insight into the importance of polyglutamate metabolism to these biological and pharmacological effects.