Summary. Hepatitis B virus (HBV) reactivation during cytotoxic chemotherapy for cancer may complicate treatment and cause liver damage. The complication has been reported to occur in 10% to over 50% of HBV carriers, but the factors that determine which patients will develop reactivation remain unclear. The objective of the study is to test the hypothesis that the prechemotherapy HBV DNA level is a risk factor for the development of HBV reactivation. We studied 41 women undergoing cytotoxic chemotherapy for breast cancer, 17 of whom developed reactivation and 24 who did not. We developed a novel, ultra-sensitive, real-time polymerase chain reaction assay for the measurement of HBV DNA. The sera of 37 patients (16 who developed reactivation and 21 who did not) were available for measurement of HBV DNA using this technique. The results showed that patients in the reactivation group had a significantly higher median HBV DNA load (1.03 × 106 copies/mL; range <2.9 × 103 to 8.723 × 107) than did the nonreactivation group (<2.9 × 103 copies/ml; range <2.9 × 103 to 6.331 × 107) (P < 0.001). The optimal cut-off between the two groups was found to be at serum HBV DNA level of 3 × 105, which gave a sensitivity of 81.0% and a specificity of 85.0%. In conclusion, for breast cancer patients receiving standard cytotoxic chemotherapy, a high HBV viral load prior to the administration of cytotoxic chemotherapy is a significant predictive factor for the development of HBV reactivation. Such information may be useful in determining which patients would benefit most from prophylactic antiviral therapy during cytotoxic chemotherapy.