The levels of endogenous serum digoxin-like immunoreactive substances were investigated during development of encephalopathy in patients with fulminant hepatic failure. The 67 patients studied had varying degrees of hepatic failure as a result of viral hepatitis or paracetamol overdose. Serum levels of digoxin-like immunoreactive substances were significantly increased in both viral hepatitis and paracetamol overdose, with mean values of 0.42 ± S.D. 0.25 ng per ml (n = 36) and 0.53 ± 0.19 ng per ml (n = 31), respectively, as compared to normal control subjects with mean values of 0.01 ± 0.02 ng per ml (n = 21, p < 0.001). A statistically significant correlation was found between serum digoxin-like immunoreactive substances and the degree of encephalopathy in the viral hepatitis patients and with the serum creatinine in the paracetamol overdose patients where renal failure was more severe. No correlation was found with liver damage as assessed by the prolongation of the prothrombin time, serum AST or bilirubin values. Experiments with ultrafiltration and heating showed that both free nonprotein-bound digoxin-like immunoreactive substances and the total digoxin-like immunoreactive substances measured were increased. Column chromatography of ultrafiltrates of fulminant hepatic failure serum on Sephadex G-25 demonstrated at least two peaks with digoxin-like immunoreactive activity. Reduced renal function is an important factor in the increased serum level of digoxin-like digoxin-like immunoreactive substances, but their presence due to liver failure, where there is increased permeability of the blood-brain barrier, could be relevant to the development of hepatic encephalopathy.