1. Alkaline phosphodiesterase I was present in rat liver at approx. 100-fold greater activity than alkaline phosphatase, and in rat bile at approx. 25-fold greater activity. 2. Rat serum alkaline phosphodiesterase I was increased 6-fold whilst serum alkaline phosphatase was increased only 2-fold 96 h after bile duct ligation. 3. In contrast to alkaline phosphatase, hepatic alkaline phosphodiesterase I was not affected by bile duct ligation, suggesting its raised serum activity was due to bile regurgitation rather than overspill of the enzyme from liver into blood. 4. Gel filtration showed that 8 and 96 h after bile duct ligation the serum contained a high molecular weight form of alkaline phosphodiesterase I. 5. It is suggested that alkaline phosphodiesterase I offers a potentially useful indicator of biliary obstruction in the rat.