Hepatitis B virus (HBV) genotype and precore/core promoter mutations have been implicated in spontaneous and interferon alfa (IFN-α)—related hepatitis B e antigen (HBeAg) seroconversion. We performed a retrospective analysis of a previously reported randomized controlled trial to determine the effects of HBV genotype and precore/core promoter mutations on IFN-α response in patients with HBeAg-positive chronic hepatitis. Clinical data and stored sera from 109 (95%) patients in the original trial were analyzed. Seventy-three patients received IFN-α and 34 received no treatment (controls). Almost all patients had HBV genotypes B (38%) and C (60%). Antiviral response was achieved in 39% and 17% of IFN-α—treated patients (P = 0.03) and in 10% and 8% of untreated controls (P = 0.88) with HBV genotype B and C, respectively. Multivariate analysis identified HBV genotype B, elevated pretreatment alanine aminotransferase (ALT) levels, and low pretreatment HBV-DNA levels but not IFN-α treatment as independent factors associated with antiviral response. Among the 66 patients with elevated pretreatment ALT level, antiviral response was achieved in 57% and 21% of IFN-α—treated patients (P = 0.019), and in 25% and 8% of untreated controls (P = 0.45) with HBV genotype B and C, respectively. Multivariate analysis showed that genotype B and low pretreatment HBV-DNA levels were independent predictors of antiviral response. In conclusion, our data showed that HBV genotype B was associated with a higher rate of IFN-induced HBeAg clearance compared with genotype C. Stratification for HBV genotypes should be considered in future clinical trials of antiviral therapy of chronic hepatitis B. (HEPATOLOGY2002;36:1425–1430).