Expression of the firefly luciferase gene under the control of viral or fish promoters was observed in fish tissue after direct DNA injection of plasmid DNA. Plasmid DNA containing the firefly luciferase gene was injected into the skeletal muscle of rainbow trout (Oncorhynchus mykiss), and levels of luciferase activity were found to be dependent on the controlling promoter and the amount of injected DNA. Plasmids using the cytomegalovirus immediate early promoter (CMV-IEP) consistently produced the highest levels of luciferase activity. Maximal activity was observed five to seven days postinjection with 50 micrograms of DNA. This activity persisted in the tissues for as long as 115 days postinjection. When the DNA was examined up to two months postinjection, the predominant form was unreplicated, unintegrated DNA in linear and relaxed circular conformation. Expression of injected DNA was found predominantly within muscle cells along the injection path and in scattered muscle cells anterior to the injection site.