Ubiquitin, which can conjugate with cellular proteins, is classified into two forms: free ubiquitin and multiubiquitin chains. The latter is active as a signal for degradation of the targeted proteins. We found both forms in human serum and, using two immunoassays, quantitated them in sera from healthy subjects and patients with some diseases. Because of putative leakage of erythrocyte ubiquitin, hemolytic serum and serum obtained after long incubation (> 1-2 h) of blood at room temperature were excluded. Serum concentrations of multiubiquitin chains and free ubiquitin were substantially higher in rheumatoid arthritis and hemodialysis patients, respectively, than healthy subjects. Additionally, in acute viral hepatitis, serum multiubiquitin chain concentrations were increased in the acute phase, decreased in the recovery phase, and correlated with alanine and aspartate aminotransferase activities (r = 0.676 and 0.610, P < 0.0001 and < 0.001, respectively). Therefore, serum ubiquitin may have prognostic value.