Hemophilia A is a sex-linked disorder that results from a deficiency of functional factor VIII and is currently treated by protein replacement therapies. Within the past decade, gene therapy efforts have come to the forefront of novel therapeutics. In this work, a dual-vector approach was employed in which recombinant adeno-associated viral (rAAV) vectors expressing the heavy and light chains of the murine factor VIII gene were delivered either intramuscularly or intravenously to a mouse model of hemophilia A. From in vitro work, it was determined that coinfection with both vectors is required as heterodimerization of the heavy and light chains occurs intracellularly. In vivo, therapeutic levels of factor VIII expression were achieved throughout the duration of the study (22 weeks). Intravenous and intramuscular delivery resulted in a maximal average expression of 31.4 +/- 6.4 and 29 +/- 6.5% of normal murine factor VIII levels, respectively. Western blots of cryoprecipitate as well as immunostaining of injection sites with an anti-murine factor VIII light chain antibody also confirmed the expression of factor VIII. Because the murine form of the gene was used in the mouse model, less than 1 Bethesda unit of inhibitors was noted. This work demonstrates the feasibility of using rAAV vectors for the long-term treatment of hemophilia A.