The following studies were done to determine the mechanism of the increse in rat liver alkaline phosphatase activity after bile duct ligation. Antiserum was raised in rabbits to highly purified rat liver alkaline phosphatase. In immune titration experiments, the 350% increase in rat liver alkaline phosphatase activity caused by bile duct ligation was paralleled by a similar increase in immuno-precipitated alkaline phosphatase protein. In a second set of experiments, rat liver alkaline phosphatase was labeled with L-[3H]leucine injected into portal veins. Alkaline phosphatase was purified by antibody affinity chromatography followed by immunoprecipitation. The incorporation of L-[3H]leucine into alkaline phosphatase was significantly higher in bile duct-ligated rats than in controls, 68,357 ± 7,144 vs. 19,297 ± 3,076 dpm per gm liver (p < 0.001) and 349 ± 36 vs. 104 ±17 dpm per gm protein (p < 0.001). In a third set of experiments, the incorporation of L-3H-amino acid into highly purified rat liver alkaline phosphatase was measured. Rat liver alkaline phosphatase was purified by means of sequential iV-butanol extraction, antibody affinity column chromatography, preparative polyacrylamide gel electrophoresis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was significantly more L-3H-amino acid incorporated into alkaline phosphatase in bile duct-ligated rats compared to sham-operated rats, 3,565 vs. 704 dpm per gm liver and 19,656 vs. 3,843 dpm/gm protein. The data suggest that bile duct ligation increases the synthesis of rat liver alkaline phosphatase.