Summary An appreciable uptake of H3-pteroylglutamate (H3PGA) by chicken liver cell-free extract was found. The uptake was dependent on NADPH and was slightly stimulated by ATP, manganese ions and mercaptoethanol. Aminopterin at low concentrations (2 × 10-3 μM) inhibited the uptake of H3PGA. The latter finding indicates the formation of a reduced folate derivative at the tetrahydrofolate level as a prerequisite for uptake. The temperature and pH optima of the reaction were studied. The possibility of utilizing the radio-assay for determination of folates in blood and tissues, by isotopic dilution, is discussed.