In view of role played by pteroylpolyglutamates as actual folate coenzymes in single-carbon unit metabolism, the effect of folic acid deficiency on the distribution of these compounds in rat liver has been studied. In deficient rats the liver levels of pteroylpolyglutamates and in particular of those containing a higher number of glutamyl residues are more decreased as compared with monoglutamates. In fact penta- and other higher polyglutamates: mono-, di- and tri-glutamates ratio is lower in deficient rats than that in control rats. The decreased amount of these coenzymic forms observed in this condition may be considered as the consequence of the severe alterations of process which adds glutamate moieties to monoglutamate forms.