The expression of naked plasmid DNA coding for firefly luciferase (pRSVluc) or a secreted protein, human-alpha-1-antitrypsin (pRcCMVhAAT) in mouse skeletal muscle was characterized following administration by an improved intramuscular injection technique. Injection guided by intense illumination along the longitudinal axis of the mouse quadriceps muscle and parallel to the myofibers yielded 200-fold higher levels of luciferase expression than perpendicular injection. Luciferase expression was inhibited by an excess of non-coding DNA or dextran sulfate suggesting that muscle DNA uptake mechanism(s) can be saturated. Injected plasmid DNA was rapidly eliminated from the muscle as evidenced by tissue distribution studies of radiolabeled hAAT plasmid and Southern analysis. However, PCR analysis demonstrated that hAAT cDNA persisted in the muscle for at least 1 month after injection. Immunohistochemistry techniques indicated that the hAAT gene was expressed by the muscle fibers. ELISA analysis of serum samples collected from intramuscularly injected mice demonstrated that secreted hAAT protein concentration peaked in serum by day 7, started to decline by day 14 and was barely detectable 21 days post-injection. RT-PCR analysis demonstrated that hAAT transcript persisted at the site of injection for at least 1 month indicating that the decline of serum hAAT concentration 21 days post-injection was not due to the absence of hAAT transcript. However, the decline of hAAT protein concentration in the serum was inversely correlated with accumulation of murine anti-hAAT antibodies in circulation.