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Objective: To determine the prevalence of hepatitis G virus (HGV) carriage in Queensland blood donors., Design: Cross-sectional survey with retrospective longitudinal study of HGV-positive donors., Setting: Brisbane Red Cross Blood Bank, 1995., Subjects: 100 consecutive blood donors attending the Blood Bank on two days in October 1995 and 20 blood donors with a raised plasma alanine aminotransferase (ALT) level on their last donation., Outcome Measures: Presence of HGV RNA by reverse transcription polymerase chain reaction (RT-PCR) in currently donated blood and in blood samples archived for up to 34 months. RT-PCR used two different reverse transcription methods and three different specific sets of primers and probes., Results: Five of the 120 blood donors were positive for HGV RNA by all RT-PCR methods (four of the 100 with normal ALT levels [4%] and one of the 20 with raised ALT levels [5%]). Retrospective testing of archived samples showed that four of these five had been persistently HGV RNA-positive for at least two years, while the fifth had been HGV RNA-negative on two donations before becoming HGV RNA-positive. No risk factors were identified for this donor., Conclusions: A relatively large number of Queensland blood donors (4%) are persistently HGV RNA-positive.