The immunological and biological properties of a liver-specific antigen (LSA) from rat liver are described. LSA gave interspecies cross-reactions with liver extracts from several mammalian species, but no reaction of complete identify was observed. Moreover, no cross-reaction was found with chicken or frog livers, thus indicating the rather late appearance of LSA in the process of evolution. Experiments with fetal and neonatal liver extracts have indicated that LSA appears late in fetal development and is always present at birth. The biological properties of LSA were explored by several independent approaches. LSAg-Ab immunoprecipitates were stained positively with carbon naphthoxycholine iodide, an indirect evidence of choline esterase activity. LSA was also found to bind bile acids, thus suggesting organic anion properties. Finally, LSA was detected in the circulation of rats with acute carbon tetrachloride- and galactosamine-induced hepatocellular injury. This LSA is immunologically distinct from hepatitis B antigen, alpha- fetoprotein, carcinoembryonic antigen and, from each of several serum and liver proteins tested.