Previously, we showed that rodent muscle has the ability to take up and express plasmid genes injected intramuscularly. This study now demonstrates that nonhuman primate muscle also has this ability to express injected plasmids. A scaled-up version of the standard large preparation of plasmid DNA allowed several tens of milligrams of CCC plasmid DNA to be relatively easily produced and administered to monkeys. After the injection of the E. coli β-galactosidase reporter gene in pRSVLac-Z, foreign gene expression was localized to both type I and type II myofibers. The luciferase reporter gene in pRSVL was used to quantify the amount of expression. The multiple implantation of plasmid DNA pellets was more efficient in expressing luciferase than the injection of DNA in normal saline. Luciferase activity persisted for at least 4 months after injection. However, the luciferase expression was considerably less than that in rodents. Preliminary studies explored why expression was less in monkeys. Of particular interest was the increased thickness of the perimysium of monkeys as compared to that in rodents. This increased connective tissue may decrease delivery of the plasmid DNA to the myofibers. Anti-nuclear or anti-DNA antibodies were not observed, even after repetitive DNA administrations, and no adverse effects were observed in any of the monkeys. Muscle is an important target tissue for the delivery and expression of exogenous genes. This study demonstrates that nonhuman primate myofibers, like rodent myofibers, have the special ability to take up and express intramuscularly injected plasmid DNA. The level of expression so far achieved suggests that this gene transfer technique would have limited clinical utility in humans. Efforts are underway to understand the mechanism of uptake in rodents and to understand why the luciferase expression is less in primates so that a rational approach to increasing expression can be achieved. The persistence of expression for at least 4 months and the apparent lack of toxicity suggests that plasmid DNA approaches for gene therapy of muscle should be pursued further.