The long-term persistence and activity of a naked plasmid DNA (pGL3-35S) containing a luc gene (reporter gene) controlled by a plant 35S CaMV promoter was studied in Atlantic salmon (Salmo salar L.) after injection. Atlantic salmon (mean weight 70 grams) were injected intramuscularly with 100 μg of plasmid DNA. Blood, different tissues and organs were sampled at different time points up to day 535 after injection. Southern blot analysis suggested the presence of extra-chromosomally open circular, linear and supercoiled topoforms of pGL3-35S at day 150 after injection. At day 536 open circular and supercoiled topoforms were detected. Luciferase activity was detected at the injection site up to 536 days post-injection of pGL3-35S, where it peaked at day 150 and decreased to approximately 17% of its maximum activity by day 536. Our study demonstrated that a plasmid containing the 35S promoter was able to induce expression of a reporter gene/protein in fish in vivo and that the plasmid DNA persisted for a prolonged time after intramuscular injection.