In this study our aim was to investigate the persistence and distribution of plasmid DNA in Atlantic salmon. Atlantic salmon (mean weight 70 g) were injected with 100 microg of plasmid DNA in 100 microl of phosphate buffered saline. The fish were reared in running fresh water at temperature 0-12 degrees C and injections were performed at 8 degrees C. After intramuscular injection, samples were obtained from blood and different tissues and organs up to day 535 after injection. We found by use of Southern blotting open circular and supercoiled plasmid DNA at the injection site and plasmid DNA fragments, assessed by real-time PCR, were detected in some of the examined tissues up to day 535. A co-persistence of luciferase transcript and activity were identified at the injection site up to day 535, however analysis of DAM methylation pattern suggested that the plasmid DNA did not replicate in vivo. Our study indicated that the plasmid DNA can persist for a prolonged time after intramuscular injection.