Background Serum HBV RNA is detectable during nucleoside/nucleotide analogue therapy as a result of unaffected RNA replicative intermediates or interrupted reverse transcription. We studied the predictive value of serum HBV RNA for initial virological response during nucleoside analogue therapy. Methods Serum HBV RNA was quantified before and at 12 and 24 weeks of lamivudine or entecavir therapy. Serum HBV DNA was measured every 4–12 weeks during treatment to define initial virological response. Results Serum HBV RNA was detectable in 21 of 52 (40%) consecutive patients with a mean of 5.2 log copies/ml (male/female 35/17, mean age of 60 years with a range of 31–82, 44% HBeAg-positive, and 26 with lamivudine and 26 with entecavir) before treatment. Serum HBV RNA level at week 12 in patients with an interval from detectable to undetectable serum HBV DNA level <16 weeks was significantly lower than those with an interval ≥16 weeks (3.8 +3.8 versus 6.6 +3.5 log copies/ ml, P=0.013). After adjustment for serum HBV DNA level at week 12, serum quantatitive HBsAg level at week 12 and pretreatment ALT level, low serum HBV RNA level at week 12 predicted a shorter interval to undetectable serum HBV DNA level (adjusted hazard ratio =0.908, 95% CI 0.829, 0.993, P=0.035). Conclusions Low serum HBV RNA level at week 12 of nucleoside analogue therapy independently predicts initial virological response in treated chronic hepatitis B patients. Serum HBV RNA levels may thus be useful for optimizing treatment of chronic hepatitis B.