The effects of pH, ascorbic acid, temperature, and time on folate release from whole blood haemolysates were investigated. Maximal yields of assayable folate were obtained within 10 minutes at room temperature in whole blood haemolysates prepared at pH 3.6 in aqueous ascorbic acid solution. When prepared over a period of two hours, haemolysates yielded maximal folate values within a much wider pH range (3.0 to 6.0). Released folate is shown to be extremely labile between pH 4.7 and 3.0 but the presence of ascorbic acid in a concentration of 25 mg% or more afforded complete protection. The possible nature of the plasma folate-releasing factor and the significance of these findings in relation to the laboratory estimation of whole blood folate are discussed.