To determine the earliest time after in vivo immunisation that the spleen could be excised and held in vitro to detect an immune response, lake trout (Salvelinus namaycush) were exposed to DNP-Ficoll or Yersinia ruckeri O antigen administered by intraperitoneal injection or by bath. The spleens were excised from the fish at selected times after immunisation, placed in vitro and held in tissue culture media at 14°C until 10 days after the in vivo immunisation. They then were analysed for an immune response by counting the numbers of plaqueforming cells (PFC). PFC were first detected in samples taken 3 days after injection with either antigen. With bath immunisation, however, PFC were first seen 6 days post-immunisation when using the Y. ruckeri O antigen and no PFC above background levels appeared in fish bathed in the DNP-Ficoll solution. On day 10 after immunisation, the spleens taken directly from immunised fish always produced more PFC than when the spleens were taken through in vitro culture. A unique feature of this method, is that an immune response to an antigen may be detected by excising the spleen and holding it in in vitro culture without necessarily holding the fish for long periods of time after antigen injection or bath. This technique also adds more information on how rapidly the bacterins or antigens are processed by fish to initiate an immune response.