The reaction of methotrexate (4-amino-4-deoxy-N¹⁰-methylpteroyl-L-glutamic acid, MTX) with diethyl L-glutamate in the presence of peptide bond-forming reagents such as N,N'-dicyclohexylcarbodiimide (DCC) or diphenylphosphoryl azide gives methotrexate γ-L-glutamate diethyl ester (1), methotrexate α-L-glutamate diethyl ester (2), and methotrexate α,γ-bis(L-glutamate tetraethyl ester) (3). The major monosubstituted product is the α-isomer 2. With excess diethyl L-glutamate the bis-adduct 3 can be made to predominate, and this compound is also accessible from 2 by reaction with a second molar equivalent of diethyl L-glutamate or via a direct mixed anhydride condensation between 4-amino-4-deoxy-N¹⁰-methylpteroic acid and tetraethyl α,γ-bis(L-glutamate). Esterification of 1 and 2 yielded the corresponding triethyl esters 4 and 5, which could also be obtained via mixed anhydride coupling from 4-amino-4-deoxy-N¹⁰-methylpteroic acid and triethyl γ-L-glutamyl-L-glutamate or triethyl α-L-glutamyl-L-glutamate, respectively. Compounds 4 and 5 were also prepared from methotrexate α-monoethyl ester and methotrexate γ-monoethyl ester by reaction with diethyl L-glutamate. TLC and dry column silica gel chromatographic evidence indicates that partial racemization of the glutamate side chain occurs in MTX when either DCC or diphenylphosphoryl azide is used as the coupling reagent. This finding is explainable on the basis that N-aroyl type substitution on the glutamate side chain of MTX favors racemization via a resonance-stabilized oxazolone intermediate.