A protocol for experimental challenge with infectious pancreatic necrosis (IPN) virus was defined with brook trout Salvelinus fontinalis as the model species. Fish were exposed by immersion for 5 h in water containing IPN virus at a concentration of 105plaque-forming units per milliliter. We propose the protocol as a standard challenge based on our studies of exposure methods and host response to virulent virus. Immersion challenge induced higher and more consistent mortality than did challenge by hyperosmotic infiltration. Challenge virus should be sequentially transferred no more than five times in cell culture because further transfers reduced the virulence of the virus. At 12°C, mortalities due to primary infection occurred 6–12 d after immersion challenge and were highest (≥70%) in fish 27–56 d old. Susceptibility to lethal infection was enhanced by nutritional stress.