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The factors associated with the outcome of sequential therapy with interferon-α (IFN-α) in order to halt nucleoside/nucleotide analog (NUC) maintenance treatment for chronic hepatitis B were analyzed.A total of 50 patients with chronic hepatitis B who underwent IFN-α sequential therapy for cessation of NUC were enrolled retrospectively. The subjects received NUC plus IFN-α for 4 weeks followed by IFN-α alone for 20 weeks. Natural IFN-α of 6-MU doses was administrated three times a week. A successful response to NUC/IFN-α sequential therapy was defined as serum hepatitis B virus (HBV) DNA below 4.0 log copies/mL, serum alanine aminotransferase (ALT) below 30 IU/L, and hepatitis B e-antigen negativity at 24 months after completing the treatment.Multivariate analysis revealed that hepatitis B surface antigen (HBsAg) of 3.0 log U/mL or more (P < 0.002) and hepatitis B core-related antigen (hepatitis B core-related antigen [HBcrAg]) of 4.5 log U/mL or more (P < 0.003) at the start of IFN-α administration were significant factors associated with a 24-month non-response. Maximal levels of ALT and HBV DNA during the follow-up period after completing IFN-α therapy were significantly related (P < 0.001), and receiver operating characteristic analysis showed that both maximal ALT (P < 0.001) and HBV DNA (P < 0.001) were significantly related to the final 24-month response.The combinational use of HBsAg and HBcrAg levels may be useful to predict the 24-month outcome of NUC/IFN-α sequential therapy. Maximal levels of ALT and HBV DNA during post-treatment follow-up may also help monitor responses to IFN-α sequential therapy.