The diagnostic value of determining total conjugated serum bile acid (SBA) concentrations was evaluated in fasting dogs with spontaneous liver disease. Conjugated primary SBA values were determined by radioimmunoassay in 12 healthy dogs, 64 dogs with hepatobiliary disease, and 9 dogs with intestinal disorders unassociated with clinical or biochemical evidence of liver disease. Reference values for SBA concentrations ranged from 0 to 5 mumol/L and were not significantly different from those determined in dogs with intestinal disease (P less than 0.05). Mean SBA concentrations determined in dogs with portosystemic shunts, glucocorticoid-induced hepatopathy, hepatic neoplasia, hepatitis, cholestasis, and cirrhosis were significantly greater than reference values (P less than 0.05). The mean SBA concentration in dogs with glucocorticoid-induced hepatopathy was significantly (P less than 0.05) lower than that in all other clinical groups of dogs with liver disease, except in dogs with cholestasis. Although these 2 groups were statistically indistinguishable, dogs with glucocorticoid-induced hepatopathy generally had lower SBA values (2 to 37 mumol/L) than did the group with cholestasis (2 to 562 mumol/L). The SBA concentrations in fasting dogs were weakly correlated with histologic evidence of hepatic damage, as determined by a total biopsy score (r = 0.28, P less than 0.02). Because total SBA concentrations were increased in 89% of all dogs with hepatobiliary disease, the determination of SBA appears to be a sensitive test of hepatic dysfunction.