The substrate specificity and kinetic properties of folylpoly-gamma-glutamate synthetases, which have been purified from several bacterial and mammalian sources, are under investigation. Pteroylpolyglutamate analogs have been synthesized and will be used to map the folate binding sites of these enzymes. Potential inhibitors of the enzymes have been synthesized, or are in preparation. Inhibitors of folylpolyglutamate synthetase will be tested for their ability to cause a cellular folate depletion by preventing the formation of pteroylpolyglutamates, the forms of the vitamin that are selectively retained by tissues. The ability of these inhibitors to increase the exogenous requirement for products of one carbon metabolism, and to potentiate any differences in nutritional requirements between tumorigenic and nontumorigenic cells, will be studied. The enzymatic conversion of antifolates to polyglutamate forms, and the effects of this metabolism on the cytotoxic action of these drugs, will be investigated.