We attempted to accumulate the basic data for evaluation of activated lymphocyte therapy for small animal medicine. The peripheral blood mononuclear cells (PBMCs) collected from healthy dogs were activated using anti-CD3 antibody and human recombinant (hr) interleukin (IL)-2 and reactivated using hr interferon (IFN)-α and hr IL-2. The property of obtained cells was compared with PBMCs. The number of cells was shown to have increased approximately > 50 -fold by cultivation. The proportion of CD8⁺ cells was significantly increased, the cytotoxicity of the cultured cells was revealed to have been reinforced. Additionally, CD56 mRNA levels tended to have increased. The cells obtained by this method were confirmed to be activated lymphocytes. Furthermore, we investigated the effects of sequential administration of the obtained cells to healthy dogs. By sequential administration of the activated lymphocytes, the cell proliferative activity, proportion of CD4⁺ cells and CD8⁺ cells, and serum IFN-γ concentration were shown to have increased, and no severe adverse effects were observed. Consequently, activated lymphocytes could be induced using anti-CD3 antibody and IL-2 in healthy dogs, and sequential administration of activated lymphocytes reinforced the recipient's immunity.