1. The recent suggestions that folate polyglutamate biosynthesis demonstrated in vivo with [3′5′9(n)-3H]folic acid is due to exchange and cannot be achieved with [2-14C]folic acid has been demonstrated to be untrue.
2. The compounds formed with both types of radioactive tracer have been shown to be folate polyglutamates from their elution position from precalibrated ion-exchange columns and their susceptibility to hydrolysis by a γ-carboxypeptidase (conjugase) from human sera.
3. By the use of the methods involving [2-14C]folic acid, reported by others to give only folate monoglutamates, we have been able to demonstrate clearly the presence of folate polyglutamates.