In chronic hepatitis B transition from active replication to viral latency (HBeAg seroconversion) usually leads to remission of the disease. alpha-Interferon (IFN) therapy induces HBeAg seroconversion in about one-third of the patients, thus leaving the majority of patients with persistent disease. Eighteen chronic hepatitis B patients who did not respond (HBeAg seroconversion and clearance of HBV-DNA) to an initial 16-week course of IFN subsequently received IFN again after at least 6 months of no therapy. The repeated therapy consisted of 1.5-5 MU lymphoblastoid IFN daily for 16 weeks. Treatment effects were monitored by quantitative measurement of HBeAg and HBV-DNA. To analyze whether the results were related to patient characteristics known to affect the response to initial treatment, a predicted response rate, based on pre-treatment factors, was determined. After a follow-up of 52 weeks, 2 of the 18 patients (11%) had responded to therapy. Two additional patients became HBV-DNA-negative with sustained HBeAg positivity. All patients remained HBsAg-positive. According to the pre-treatment parameters, a response was predicted for 9 of the 18 patients (50%). This predicted response rate was significantly higher than the actual response rate (p = 0.03). In conclusion, this pilot study with moderate dosages of IFN suggests that the HBeAg seroconversion rate after repeated IFN treatment is low for previous non-responders and probably is not related to important clinical characteristics that influence the response to initial IFN treatment. A large controlled trial with higher doses of IFN is desirable to further evaluate the benefits of retreatment.