Injection of plasmid DNA encoding antigens into rodents followed by electroporation improved the immune response when compared with injection without electroporation (Widera et al. J Immunol 2000;164:4635-40; Zucchelli et al. J Virol 2000;74:11598-607; Kadowaki et al. Vaccine 2000;18:2779-88). The present study describes the extension of this technology to farm animals, by injecting plasmid DNA encoding mycobacterial antigens (MPB70, Ag85B and Hsp65) into the muscles of goats and cattle using two different types of electrodes, both allowing DNA delivery at the site of electroporation. The animals were vaccinated under local anaesthesia without any observed immediate or long-term distress or discomfort, or any behavioural signs of muscle damage or pathological changes after the electroporation. DNA-injected and electroporated goats showed increased humoral response after the primary vaccination when compared with nonelectroporated animals. Improved T-cell responses following electroporation were observed in hsp65 DNA-vaccinated cattle. DNA injection with or without electroporation did not compromise the specificity of the tuberculin skin test. In conclusion, a protocol applying in vivo electroporation free of side effects to farmed ruminants was established. In addition, we show that DNA vaccination in combination with electroporation can improve the primary immune responses to the encoded antigens.