In this study, rabbits were used as a model for gene therapy for hemophilia B. Human factor IX cDNA was transferred to cultured normal rabbit skin fibroblasts (RSF) by a recombinant plasmid (pCMVIX) or retrovirus (XL-IX or N2CMVIX) constructed in our laboratory. Infected fibroblasts capable of synthesizing and secreting high levels of biologically active human factor IX protein were selected and embedded in a collagen matrix. The latter was surgically implanted into rabbits as autografts or allografts. Human factor IX protein was detected in the plasma of all the grafted rabbits, and its expression has been maintained for more than 10 months at the time of writing. In addition, we have improved and simplified the method of implantation from surgically grafting the tissue-like matrix to the injection of the infected cell-collagen mixture subcutaneously. Using the latter method, human factor IX in rabbits injected with RSF-N2CMVIX reached a peak of 480 ng/ml plasma, and its expression has continued for more than 3 months at the time of writing. We suggest that the simplified method of transplantation by subcutaneous injection would offer an effective and acceptable approach to somatic cell gene therapy and may be practical for human trials.