Administration of interleukin 2 (IL-2) has been associated with potent in vitro antitumor effects. However, systemic in vivo toxicity has been problematic. Because local delivery and liposomal formulations of IL-2 may improve the therapeutic index, we used dogs to evaluate and compare immunological activation of inhaled free IL-2 and IL-2 liposomes. Twelve normal dogs were treated with nebulized IL-2 formulations and controls for 2 to 7 weeks. Cellular immune activation of peripheral blood mononuclear cells and bronchoalveolar lavage (BAL) effector leukocytes against tumor cell lines, changes in effector leukocyte populations, and toxicity were monitored. No toxicity was seen with either aerosolized free IL-2 or IL-2 liposomes. Free IL-2 given at 0.5 x 10(6) Biologic Response Modifier Program (BRMP) units twice daily to dogs resulted in increased peripheral blood mononuclear cell activation compared with saline control-treated dogs. IL-2 liposomes given at 0.5 x 10(6) BRMP units twice daily to dogs resulted in significantly increased BAL effector activation compared with IL-2 liposomes given at 1.0 x 10(6) BRMP units once daily (P = 0.018) and empty liposome controls (P = 0.016). The BAL leukocyte cell count was increased significantly after inhalation of IL-2 liposomes versus inhalation of free IL-2 (P = 0.011). BAL effector populations included a greater proportion and total number of lymphocytes and eosinophils after treatment with IL-2 liposomes. Nontoxic activation of pulmonary immune effectors for the treatment of cancer in the lung may be possible using nebulized IL-2 liposomes.