A novel non-metabolic role is proposed for dihydropteroyl hexaglutamate as a critical link binding together sub-structures of the tail of Escherichia coli bacteriophage T4. Six molecules of this folate compound have been found to be components of the complex tail baseplate of the phage particle. The baseplate is assembled using a total of at least 18 viral gene products in a series of reactions in which six wedge-like elements (each 0.7 X 10(6) daltons) bind symmetrically around a central tail plug (1.55 X 10(6) daltons) to form a flat hexagonal structure. It appears likely that the pteridine portion of the folate binds to a site on a viral-induced dihydrofolate reductase molecule, a wedge component, while the glutamate residues of the folate bind to a viral-induced thymidylate synthase molecule, a central plug component. Additionally, it appears that the folyl glutamate residues play a role in forming a flexible bond between the proximal end of the phage long tail fiber and the baseplate. Two bacteriophages attacking a quite different bacterial host, Pseudomonas syringae, have been isolated and partially characterized. Both phage strains have tail structures morphologically analogous to T4. Both were irreversibly inactivated by an enzyme which cleaves the gamma-glutamyl bonds of folyl polyglutamate. It appears that these Pseudomonas phage particles also contain a folyl poly-glutamate whose integrity is essential for their infectivity.