The liver is one of the prime targets for gene therapy, and the correction of defects in a variety of clotting factor genes is one of the main goals of liver-directed therapies. The use of transcriptional regulatory elements derived from these genes may provide for the optimal expression of transduced genes. We have applied our knowledge of the promoter structure of the clotting Factor IX gene to design optimized expression vectors for use in gene therapy. The activity of the proximal promoter has been augmented by the introduction of a multimerized upstream site which we have previously shown to be a prime regulator of the pro- moter. Introduction of this element increases promoter activity at least 20-fold over the proximal promoter alone when assayed in the human liver cell line Hep G2. This optimized promoter is significantly more active than the SV40 enhancer/early promoter. The expression of the optimized Factor IX promoter is also more persistent in the short term. The inclusion of a liver-specific locus control region, derived from the apolipoprotein E/C locus, did not further augment expression levels. These Factor IX vectors also exhibit a high degree of tissue specificity, as measured by transfection into breast and muscle cell lines.