The diastereoisomers of 5,10-methylene 5,6,7,8-tetrahydropteroyl-D-glutamate were resolved and tested as substrates and inhibitors of Lactobacillus casei thymidylate synthetase. No activity was observed. The compounds were neither growth factors nor inhibitors for Lactobacillus casei, Streptococcus faecium, or Pediococcus cerevisiae. 7,8-Dihydropteroyl-D-glutamate is 50% as active as 7,8-dihydropteroyl-L-glutamate (dihydrofolate) as a substrate for L. casei dihydrofolate reductase.