DNA vaccines encoding the viral glycoproteins of viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV) have proved highly efficient in rainbow trout (Oncorhynchus mykiss) under experimental conditions. Non-specific as well as specific immune mechanisms seem to be activated. Temperature is an important external parameter affecting the immune response in fish. The present study aimed at determining the effectiveness of a DNA vaccine against VHS at different temperatures. Rainbow trout fingerlings acclimated at 5 °C, 10 °C or 15 °C, were given an intramuscular injection of 1 μg purified plasmid DNA and challenged with virulent VHSV 8 or 36–40 days later. The vaccine protected the fish well at all three temperatures, but the involvement of innate and adaptive mechanisms differed: at low temperature, non-specific protection lasted longer and at 36 dpv fish kept at 5 °C had no detectable response of neutralizing antibodies while 67% of the fish kept at 15 °C had seroconverted. Induction of Mx as measured in liver samples was delayed at 5 °C with no detectable response 7 dpv whereas fish maintained at 10 °C had significantly elevated levels of Mx3-transcripts at that time point. Immunohistochemical studies of the injection site of vaccinated fish also showed a clear effect of temperature: in fish maintained at 15 °C the vhsG-protein appeared earlier on the surface of transfected myocytes and the inflammatory response clearing away these myocytes arose earlier compared to fish kept at the lower temperatures of 5 and 10 °C. Vaccine. 2009 Jun 1227(29):3870-80. Epub 2009 Apr 23. Lorenzen, E.Einer-Jensen, K.Rasmussen, J. S.Kjaer, T. E.Collet, B.Secombes, C. J.Lorenzen, N. Department of Fish, National Veterinary Institute, Technical University of Denmark, DK-8200 Arhus N, Denmark. ello@vet.dtu.dk.