The dissociation constants of tetrahydropteroylpolyglutamates, having from one to six glutamate residues, have been determined for the two mitochondrial enzymes serine hydroxymethyltransferase and dimethylglycine dehydrogenase. The ratios of the dissociation constants for the mono- and hexaglutamate forms of the coenzyme were 200 and less than 10 for serine hydroxymethyltransferase and dimethylglycine dehydrogenase, respectively. Km and kcat values were determined for the reversible interconversion of serine and glycine as a function of the number of glutamyl residues on the coenzyme. The values in the serine to glycine direction did not significantly change with the number of glutamyl residues, but in the glycine to serine direction, there was a 9-fold increase in the kcat/Km when the longer chain polyglutamates were used as the coenzyme substrate. A sensitive and rapid method for determining the dissociation constants of proteins which bind either tetrahydropteroylpolyglutamates or their 5-methyl and 5-formyl conjugates is described.