Derivatives of folic acid occur in nature predominantly as poly (gamma-glutamyl) derivatives containing 2-8 glutamate residues. The data regarding the function of these derivatives, and their biosynthesis by eucaryotic and procaryotic folylpolyglutamate synthetases, is reviewed. The most universal functions of folylpolyglutamates appear to be (a) as the actual cofactors in vivo for folate dependent enzymes, (b) as inhibitors of folate dependent enzymes for which they are not substrates, and (c) to increase retention of folates after they are transported into cells as monoglutamates. Folylpolyglutamates also have numerous specialized functions in specific organisms, e.g. as structural components of some coliphage, and as allosteric regulators in Neurospora crassa. A single enzyme appears responsible for synthesis of all polyglutamate derivatives, regardless of length. With the recent introduction of sensitive assays this folylpolyglutamate synthetase has begun to be characterized. Although procaryotic and eucaryotic synthetases have many dissimilar properties, both types catalyze the ATP-dependent addition of L-glutamate to the gamma-carboxyl of the glutamate present in the folate. Both types also require a monovalent cation and relatively high pH. The most significant differences between the two types are in their folate substrate specificity and the product lengths derived from various folates.