The properties of folic acid gamma-glutamyl hydrolase (conjugase) [EC 22.214.171.124] in rat bile and plasma were investigated. Conjugase activity in bile showed two pH optima; one at pH 4.5-5.0 and one at pH 6.7-7.5. The enzyme activity in plasma had a broad pH--profile with an optimum at pH 6.2-7.5. Conjugase activity from both bile and plasma was inhibited in the presence of the sulfhydryl reagent, p-hydroxymercuriphenylsulfonate, and stimulated in the presence of 2-mercaptoethanol. Conjugase activity in bile was inhibited by Zn2+ at pH 7.5 but not at pH 4.5 and was much more stable to heat at pH 4.5. No separation of the biliary conjugase activity measured at the two different pH values was obtained by Sephadex G-150 chromatography. Secretion of biliary conjugase was essentially constant over a 6-hour period when activity was assayed at pH 4.5 or pH 7.5. The enzyme in bile converted pteroyltriglutamate to a mixture of about 5% glutamate an 95% gamma-glutamylglutamate at either pH, whereas the enzyme in plasma produced 23% glutamate and 77% gamma-glutamylglutamate. The possible contribution of biliary conjugase to intestinal absorption of folate polyglutamates is discussed.