We aimed to investigate the incidence of occult hepatitis B infection (OBI) in patients with “cryptogenic” hepatocellular carcinoma (HCC) and to study the HBV replicative activity in these patients. Tumorous and adjacent nontumorous liver tissues were obtained from 33 cryptogenic HCC patients and 28 HCC patients with identifiable causes (13 with chronic hepatitis B [CHB], six with chronic hepatitis C, and nine alcohol-related). OBI was identified by nested polymerase chain reaction (PCR). Intrahepatic HBV DNA, covalently closed circular DNA (cccDNA), and pregenomic RNA (pgRNA) were quantified by real-time PCR and reverse-transcription PCR (RT-PCR), respectively. OBI was identified in 24 (73%) cryptogenic HCC patients, one (17%) HCC patient with HCV, and five (56%) patients with alcohol-related HCC. In HCC patients with OBI, HBV DNA were detected in ≥2 HBV genomic regions more often in nontumorous tissues than in tumorous tissues (90% versus 57%, respectively; P = 0.007). Cryptogenic HCC patients with OBI had lower intrahepatic total HBV DNA levels than HCC patients with CHB (median: 0.010 versus 3.19 copies/cell, respectively; P < 0.0001). Only six (26%) cryptogenic HCC patients with OBI had detectable cccDNA (median: <0.0002 copies/cell), which was significantly lower than that of the CHB patients (median: 0.005 copies/cell; P < 0.0001). HBV pgRNA were detectable in 12 (52%) cryptogenic HCC patients with OBI (median: 0.0001 copies/cell), which was significantly lower than that of the CHB patients (median: 2.90 copies/cell; P < 0.001). Conclusion: 73% of patients with apparently unidentifiable causes for HCC were HBV-related. The detection rate was higher in nontumorous tissues than tumorous tissues. The low intrahepatic HBV DNA and pgRNA levels indicated that persistent viral replication and possibly HBV integration are the likely causes of HCC in OBI patients. (HEPATOLOGY 2011;)