1. It has been demonstrated that the hydrodynamics-based procedure has high efficiency to deliver foreign genes into the liver. The widespread use of this procedure in gene function studies and as a treatment option for liver and other organ diseases puts considerable importance on the investigation of various conditions that affect hydrodynamics-based gene delivery into mouse liver in vivo. 2. Various conditions, including the volume, speed and solution of the injection and the state, gender and strain of the animal were manipulated to evaluate their effect on the expression levels in mice of human factor IX (hFIX) 8 h after tail vein injection of the plasmid pCMV-hFIX. 3. It was found that an injection volume of 2-2.5 mL and an injection speed of 5-7 s were very effective in delivering DNA into the mouse liver. Using Ringer's solution as an injection fluid increased the efficiency of hFIX expression. 4. Anaesthetized mice expressed higher hFIX than conscious mice. Males expressed higher hFIX than females. The ICR mouse strain demonstrated higher expression of the foreign gene than did the C57 strain. 5. The effects of these specific factors on hFIX expression may be caused by variations in hydrostatic pressure, the degree of liver damage and liver size. 6. It can be concluded that there are optimal conditions for hFIX expression in the liver. This information may be helpful for the application of hydrodynamics-based procedures in the investigation of gene expression and gene therapy.