In an effort to gain knowledge on immune functions in beluga whales, Delphinapterus leucas, we have used two physical methods for the purification of T lymphocytes of spleen cells. Isolation by sheep red blood cells (SRBC) rosetting and by adherence on nylon wool columns were tested. SRBC-rosetting gave unreliable results in obtaining purified T cells. Therefore, the purification of T cells was done using nylon wool columns. Less than 3% of the IgM(+) B cells remained in effluent populations. In the later population, 45% gave positive staining with mouse anti-human CD4 allowing us to verify functionality of the cells. The study of calcium mobilization and tyrosine kinase activation, mediated by CD4 cross-linking permitted verification of the functionality of cells. We also showed that upon activation with mitogens, beluga T cells upregulate the density of MHC class II molecules on their surfaces. CD4 cross-linking with a specific antibody inhibited the proliferation response. Overall, the activation of beluga whales lymphocytes did not differ markedly from what is known in other species. This study can help in the groundwork for functional investigation of the beluga whale's immune system.