We previously demonstrated that rAAV vectors carrying human and canine factor IX (FIX) cDNA can infect, stably persist, and secrete functional human and canine FIX following direct intramuscular injection. In an attempt to improve FIX protein secretion for eventual therapeutic use, we set out to determine if alteration of the AAV capsid would affect skeletal muscle transduction and factor IX secretion. Two reasons to pursue this question were (1) the persistence of high-titer neutralizing antibody (NAB) to AAV2 and (2) our previous study that supported a restricted tropism of muscle fiber types to AAV2 transduction. Using an identical CMV/canine factor IX (cFIX) expression cassette, we cross-packaged this genome into virions generated from each of the five AAV serotypes. In a dose-response assay, equivalent amounts of rAAV/cFIX serotypes were tested in vitro and in vivo. In tissue culture cells, FIX antigen levels secreted into the supernatant varied depending on the AAV serotype used; type 2 transduced maximally, with serotypes 3, 1, 5, and 4, respectively, expressing lower levels. However, when the same viruses were tested in vivo using immunodeficient NOD/SCID animals, we obtained surprisingly different results. While the time to onset of detectable serum levels appeared the same for all serotypes, types 1, 3, and 5 produced 100- to 1000-fold more cFIX than type 2. In fact, 12 weeks after transduction, type 1 continued to express levels of cFIX on average at 80 microg/ml followed by type 5 (6.52 microg/ml), type 3 (3.27 microg/ml), type 4 (258 ng/ml), and finally type 2 (90 ng/ml). Coagulant activity of cFIX as measured by aPTT supported the circulating levels measured by ELISA demonstrating the secreted protein was functional, and RT-PCR of injected tissue correlated with the serotype-specific transduction data. In summary, we found significant differences in cFIX expression upon introducing various rAAV serotypes into mouse muscle. These data have direct bearing on the design of AAV gene therapy clinical trials for hemophilia and should also extend to most therapeutic transgenes.