We describe a methodology to obtain from the trout T-lymphocyte-like cell cultures showing in vitro antigen-dependent cell (ADC) proliferation. ADC cultures were developed from each of 3 outbred rainbow trout that survived two consecutive viral haemorrhagic septicaemia virus (VHSV) infections (an important salmonid fish disease in Europe). As stimulating antigen, we used a yeast recombinant form of the glycoprotein G (G4) of VHSV. No similar ADC cultures could be obtained from non-infected control trout kidney cells incubated in the presence of G4, confirming previously published observations. For long-term culture, and to overcome the absence of inbred trout populations, autologous haematopoietic adherent (Ad) cells were obtained and maintained from each individual trout kidney and periodically used as viral antigen-presenting cells (called G4-pulsed Ad cells) to autologous ADC cultures. The methodology to obtain trout ADC lines constitutes a new tool that would facilitate further studies on the comparative and developmental immunology of lower vertebrates. It could also be applied to research on fish vaccination and viral antigen-presentation mechanisms.