Tetrahydropteroylpolyglutamates containing up to seven Glu residues were tested as substrates for Lactobacillus casei thymidylate synthase. The Km values decreased from 24 microM for the monoglutamate to 1.8 microM for the triglutamate. Addition of residues 4, 5, 6, and 7 did not decrease the Km further. When monoglutamate and polyglutamate substrates were simultaneously incubated with the enzyme, the rate observed was characteristic of the polyglutamate even when the monoglutamate concentration was 44 times that of the polyglutamate. Iodoacetamide treatment inhibited the enzyme to the same extent with monoglutamate and polyglutamate substrates. Addition of 0.3 M NaCl doubled the rate obtained with the polyglutamate substrate whereas the rate with the monoglutamate was inhibited 25%. MgCl2 stimulated the reaction only 10% with the polyglutamate substrate compared with 80% stimulation obtained with the monoglutamate. Inhibition by fluorodeoxyuridylate was similar with both mono- and polyglutamate substrates; however, with the phosphonate derivative of fluorodeoxyuridine, the polyglutamate substrate enhanced inhibition 5- to 8-fold.