Background In vivo adeno-associated virus (AAV) delivery to adult liver results in sustained expression of the transgene. However, it has been suggested that AAV delivery to the newborn liver may result in transient expression. In the present study, we analysed transgene expression after AAV8 delivery of a therapeutic or a marker gene to newborn rat liver. Methods Recombinant AAV 8 vectors carrying either the human UGT1A1 cDNA or the lacZ gene were injected intravenously in 2-day-old Gunn or Wistar rats. Serum bilirubin level was recorded in Gunn rats and beta-galactosidase expression was monitored by immunohistochemistry or enzyme activity. The molecular forms of AAV genome were analysed by the polymerase chain reaction and Southern blotting in whole liver and by the quantitative polymerase chain reaction in macroscopically dissected beta-galactosidase clusters. Results In Gunn rat, complete serum bilirubin normalization occurred after AAV delivery but hyperbilirubinemia resumed thereafter. Similarly, beta-galactosidase expression was maximum at day 7, but only a few (less than 1%) beta-galactosidase positive cells were recorded at 1 or 3 months. These cells gathered in small clusters and the AAV copy number was 75-fold higher in positive cell clusters than in the surrounding parenchyma. Conclusions The results obtained in the present study show that in vivo AAV delivery to newborn rats results in transient expression in most hepatocytes. Expression of the trangene was persistent in small clusters of cells and preliminary data support the hypothesis that integration of the viral genome occurs in these clusters. Altogether, our data confirm the low efficiency of AAV vectors for gene therapy of liver diseases when delivered in the newborn period. Copyright (C) 2009 John Wiley & Sons, Ltd.