Atlantic halibut Hippoglossus hippoglossus, age 8 mo and weighing 20 g, were challenged by either intraperitoneal injection (i.p.) or by bath exposure using nodavirus isolated from Atlantic halibut. Fish were sampled at intervals over a 41 d period, starting on Day 5 post-challenge. Although no clinical disease or mortality was recorded, the data show that nodavirus did successfully propagate in i.p.-challenged fish. Using conventional end-point reverse transcription (RT)-PCR, nodavirus was detected in the kidney of all examined i.p.-challenged fish, and further in the head, heart, liver and posterior intestine of most of these individuals. Quantitative real-time RT-PCR revealed that the amount of virus in head samples from the i.p.-challenged group increased during the experiment. The presence of nodavirus in nervous tissue of i.p.-challenged fish was detected by immunohistochemistry from Day 13 post-challenge. In the retina, virus positive cells were found adjacent to the circumferential germinal zone at the ciliary margin towards the iris. In the brain, a few positive cells were detected in the tectum opticum. An ELISA was developed to detect anti-nodavirus activity in plasma. The method included an optimized coating procedure, which allowed the use of non-purified nodavirus as the coating antigen in a simple indirect ELISA. An anti-nodavirus antibody response was detected from Day 19 post-challenge in i.p.-challenged fish, while a response was not detected in the bath-challenged or control fish. This experiment demonstrates a subclinical nodavirus infection in Atlantic halibut at a post-juvenile stage induced by i.p. injection of virus.