The role of the alpha-carboxyl group in methotrexate (MeAPA-Glu) and the gamma-glutamate derivative of methotrexate (MeAPA-Glu-Glu) in the reaction catalyzed by folylpolyglutamate synthetase (FPGS) has been investigated. MeAPA-Glu and MeAPA-Glu-Glu were accepted as substrates by the same FPGS species contained in an (NH4)2SO4 precipitate of mouse liver protein, as judged by a lack of additivity of product formation at saturating concentrations of both substrates. MeAPA-Gaba, the MeAPA-Glu analogue lacking an alpha-carboxyl, was inactive as a substrate for this enzyme as was MeAPA-Glu-Gaba, the analogue of MeAPA-Glu-Glu that lacked the alpha-carboxyl of the terminal glutamic acid. However, MeAPA-Gaba-Glu, the analogue of MeAPA-Glu-Glu without an alpha-carboxyl on the first glutamic acid, had activity as a substrate for FPGS that approached that of MeAPA-Glu-Glu. These results suggest that the alpha-carboxyl is essential for the binding of folyl monoglutamates to FPGS in the correct orientation to allow catalysis. Moreover, the binding of the terminal alpha-carboxyl of folyl oligoglutamates to the same residue(s) responsible for the binding of the alpha-carboxyl of folyl monoglutamates would allow correct positioning of the terminal gamma-carboxyl of the chain for reaction. This binding mechanism would be compatible with the utilization of a single enzyme species for the addition of glutamate to the monoglutamate or oligoglutamate forms of folates and folate analogues.