Abstract Background: Hepatitis B virus (HBV) DNA often remains detectable in serum despite clinical recovery and loss of HBsAg. Objective: To study whether coinfection with HIV and HCV influence the chance of detecting HBV DNA in sera with markers of past hepatitis B. Study design and results: The test panel included 160 anti-HBc-positive/HBsAg-negative sera collected in the diagnostic setting. The following parameters were determined in the sera: anti-HIV (32% positive), anti-HCV (34% positive), HCV RNA (18% positive), and anti-HBs (37% positive). A highly sensitive PCR (90%-detection limit 100 copies/ml) amplifying the terminal protein (TP) region of HBV was established and HBV DNA was detected in 12.5% of the samples. In 70% of these samples, the HBV DNA concentration was below 500 copies/ml as measured by real-time PCR in the S gene. Logistic regression analysis revealed that the chance of detecting HBV DNA was increased by a positive HCV serostatus (odds ratio 5.0, 95%-CI 1.6–15.7), whereas HIV coinfection (odds ratio 2.0, 95%-CI 0.7–5.8), anti-HBs (odds ratio 0.9, 95%-CI 0.3–2.6), and HCV RNA status (odds ratio 0.4, 95%-CI 0.1–1.7) had no statistically significant influence. In contrast, the chance of detecting HCV RNA in the subgroup of anti-HCV-positive sera was increased by HIV coinfection (odds ratio 4.5, 95%-CI 1.2–17.4). Sequencing of the TP PCR products revealed neither a specific phylogenetic origin of the circulating HBV DNA nor clustering of uncommon mutations in the TP region. Conclusions: The prevalence of HBV DNA in serum of anti-HBc-positive/HBsAg-negative subjects correlates with HCV rather than HIV serostatus.