In veterinary vaccinology, DNA-vaccines encoding the viral glycoproteins of viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV) have proved highly efficient in fish under experimental conditions. In the early phase following vaccination, innate cross-protective mechanisms are dominating but the protection becomes highly specific within 3–4 weeks at 12–15 C. Temperature is known as an important external parameter affecting the immune response in fish and the present study aimed at characterizing temperature effects on the immune response to a VHS DNA vaccine. Rainbow trout fingerlings acclimated at 5, 10 or 15 C, were given an intramuscular injection of 1 lg purified plasmid DNA and challenged with virulent VHSV 9 or 36–40 days later. The vaccine protected the fish well at all three temperatures, however the non-specific mechanisms lasted for a longer period of time at lower temperatures, hereby apparently compensating for a delayed adaptive response. At 36 dpv fish kept at 5 C thus had no detectable response of neutralising antibodies while 67% of the fish kept at 15 C had seroconverted. Immunohistochemical time-course studies of the injection site of vaccinated fish also showed a clear effect of temperature: in fish maintained at 15 C the vhsG-protein occurred earlier on the surface of transfected myocytes than in fish kept at the lower temperatures and the inflammatory response clearing away these myocytes similarly arose earlier at high temperature. Long persistence of transfected myocytes expressing the vaccine antigen might explain the prolonged stimulation of innate protective mechanisms at low temperature. Quantitative gene expression profiles suggested interferon related mechanisms as the explanation for the early protection and also supported their temperature dependent kinetics.