During the past few years great efforts have been made to construct and to test human factor VIII (hFVIII) and IX (hFIX) vectors suitable for haemophilia gene therapy in vivo. However, little is known about the molecular mechanisms of persistence and shut-off of transgene expression in the target organs after gene transfer using recombinant adenoviral vectors. To evaluate low transgene mRNA levels in different tissues, especially at long times after the gene transfer, the common northern blot method is often not sensitive enough. For this reason we developed a new, highly sensitive and species-specific method for hFIX mRNA quantification and employed it in mice treated with an adenoviral vector (Ad5CMVFIX) expressing human FIX. In addition to its very high sensitivity (lowest detection level=1 fg RNA), the method was shown to be strictly species-specific, since hFIX mRNA signals were never detected in untreated mice. In a long-term study of 18 vector-treated mice we compared the human FIX:Ag levels in the mouse plasma, the human FIX mRNA levels and human FIX vector DNA concentrations in the mouse liver. We found that a slow but continuous decrease of hFIX:Ag levels in mouse plasma was associated with a corresponding decrease of hFIX mRNA levels in the liver. However, the Ad5CMVFIX vector DNA levels did not decrease to a comparable degree, suggesting that the decrease of human FIX:Ag levels in mouse plasma is, to a significant extent, also caused by CMV promotor shut-off and only to a minor degree by loss of vector DNA.