The formation of folylpolyglutamate derivatives by germinating pea seeds (Pisum sativumL.cvHomesteader) was examined in vivo and in vitro. Differential microbiological assay of cotyledon extracts showed that total folate concentrations increased from 163 ng folate equivalents per g fresh weight at day 1 to 680 ng per g fresh weight at day 3 of germination. Over a 7 day germination period, folylpolyglutamate derivatives accounted for 46–73of the total cotyledonary folate pool. The concentration of these polyglutamate forms of folate increased 6.5 fold during the first four days of germination and then remained relatively constant.Dialyzed extracts of 1–4 day old cotyledons had ability to incorporate 3Hglutamate and 14Ctetrahydrofolate into folylpolyglutamates. This activity was mainly associated with protein precipitating at 35–45of saturation with ammonium sulphate. The folylpolyglutamate synthetase of pea cotyledons had requirements for ATP and the monoglutamate of tetrahydrofolate. The latter folate was a more effective substrate than 5,10-methylenetetrahydrofolate but the diglutamate of unsubstituted tetrahydrofolate was not utilized. Ion exchange chromatography of the reaction products suggested that 3Hglutamate and 14Ctetrahydrofolate were incorporated into di-, and tetraglutamates of tetrahydrofolate. Folates of longer glutamyl chain lengths were only detected when the synthetase reaction proceeded for 12 h or l
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