Aim: In this study, we investigated the presence of B19V in liver tissues from patients with acute liver failure (ALF) and evaluated the viral activity in infected liver. Methods: Serum and liver samples from 30 patients who underwent liver transplantation for ALF were investigated for B19V infection by real-time PCR, serological tests and examination of B19V mRNA (transcript) expression in the liver. Results: The serum and liver samples from seven patients were B19V DNA positive (103–105 copies/ml). Most of them presented detectable anti-B19V IgG, indicating persistent infection. B19V mRNA was detected in all patients, demonstrating intra-hepatic replication. Conclusion: B19V infection of the liver during the course of non-A-E ALF suggested a role of B19V, which produced the worst outcome in co-infected patients and in patients with cryptogenic ALF, in liver damage.