A sensitive and specific radioimmunoassay for rat and human serum ligandin was developed using antisera generated in rabbits by the injection of ligandin purified from liver. Because rat ligandin and human ligandin have no cross reactivity, radioimmunoassay systems for each of the serum ligandins were prepared. Highly purified ligandin labeled with125I and eluted through Sephadex G-75 column was used as a tracer. Separation of free125I-ligandin from antibody bound125I-ligandin was carried out using double antibody method. Optimal assay conditions were determined and a dose-displacement curve was obtained. Non-specific effects of serum on the assay system were excluded by one-peak appearance of ligandin in serum which was eluted on Sephadex G-75 column. The intra-assay coefficient of correlation was 0.87 (p<0.01), and the inter-assay coefficient of correlation was 0.998 (p<0.001). The serum ligandin concentrations of seven control rats were 141±61 (mean ± S.D.) ng per ml. Rat liver injury by carbon tetrachloride (CC14) caused an increase of serum ligandin associated with an almost parallel increase of serum glutamic pyruvic transaminase (S-GPT). The normal value of human serum ligandin was 5.9±4.6 ng per ml, and higher values were obtained in various liver diseases, especially at their deteriorated phase, with the possible exception of liver cirrhosis. Serum ligandin concentrations and S-GPT activities correlated significantly in acute and chronic hepatitis, but did not correlate in liver cirrhosis. In patients with constitutional hyperbilirubinemia, almost all of the serum ligandin values were within normal range. The present results suggest that serum ligandin levels may reflect liver cell damage.