When rainbow trout Oncorhynchus mykiss (mean weight, 1 g) were injected with 50200 μL of pooled serum from juveniles (IHNV neutralizing titer, 1,280) or adults (titer, 640) that had experienced natural infection with infectious hematopoietic necrosis virus (IHNV) and had survived, they exhibited relative protection of 63–100% against IHNV, compared with rainbow trout injected with control or normal serum (titer, <20) or saline. (Antibody titers, determined by plaque assay, are reported as the reciprocal of the highest serum dilution that resulted in a 50% reduction in the average number of plaques detected in negative controls.) Protection against IHNV resulted from the passive transfer of specific anti‐IHNV antibodies. Comparison with nonimmunized controls showed that passive immunization with sera obtained from individual rainbow trout (titer, 160) that had survived natural infection provided relative protection of 52–79% against subsequent challenge with IHNV. In vitro and in vivo tests with serum that‐contained anti‐IHNV antibodies produced against a single IHNV isolate indicated good cross‐protection could be attained against other IHNV isolates from Idaho. These isolates exhibited epitope differences detected with monoclonal and polyclonal antibodies. Fish (mean weight, 16 g) exposed to waterborne IHNV that resulted in a 50% cumulative mortality exhibited low prevalence and concentrations of neutralizing antibodies 1 week later. However, by 6 weeks after infection, 59% (13/22) of the fish tested had titers of 2,560. These results suggest that rainbow trout are capable of a humoral response to infection of waterborne IHNV and that anti‐IHNV neutralizing antibodies provide protection after passive transfer. Western immunoblots of sera from adult rainbow trout exhibited different reactivity patterns with IHNV proteins and did not correlate with antibody titer. The two proteins most frequently recognized were the membrane protein (M1) and the glycoprotein (G).