Several a- and y-monoesters of methotrexate (MTX) were synthesized chemically and evaluated as inhibitors of cultured human lymphoblastic leukemia (CCRF-CEM) cells and purified dihydrofolate reductase (DHFR) from rabbit liver. Chemical methods included direct HC1-catalyzed half-esterification of MTX, partial cleavage of methotrexate diesters in the presence of base, and mixed anhydride coupling from 4-amino-4-deoxy-No-methylpteroic acid. ID50 values obtained for methotrexate y-monobutyl ester against CCRF-CEM cells and rabbit liver DHFR were 0.76 X lo4 and 1.7 X lo-' mol/L, respectively. In vitro incubation of methotrexate dibutyl ester in whole human serum at 37 "C for 48 h produced only 12% cleavage to monobutyl esters and <1% cleavage to free MTX, in contrast to similar incubation in mouse serum which gave 93% free MTX. HPLC analysis of the monobutyl ester fraction from serum incubation revealed a ?/a isomer ratio of approximately 85:15, indicating that serum esterase cleavage is regioselective. The results of this study suggest that methotrexate monoesters may have a significant role in the pharmacology of methotrexate diesters in nonrodent species and should be viewed as potential therapeutic agents on their own merit.