Natural killer (NK) cells spontaneously lyse a variety of tumor cells in vitro, and are believed to play an important role in host resistance to tumor growth and metastasis in vivo. As part of our work in comparative oncology, we have designed and validated a canine NK cell assay. Of several lymphocyte isolation techniques evaluated, sedimentation of whole blood through a two-step Ficoll/Hypaque gradient (sp. gr. 1.066/1.119) followed by plastic adherence of monocytes resulted in the most pure lymphocyte population (> 95% lymphocytes). Of four cell lines evaluated as targets in the NK assay, a canine thyroid adenocarcinoma (CTAC) cell line was determined to be most sensitive, and a lymphoblastoid (CT45-S) cell line was determined to be most resistant to NK lysis. A 15 h effector-target incubation period using these targets resulted in reproducible measurement of cell specific lytic activity. Passage of canine lymphocytes through nylon wool columns did not result in a significant increase in NK activity. A final sedimentation of purified lymphocytes through a 45/50% Percoll gradient concentrated NK activity into a single band of lymphocytes. Lymphocytes forming conjugates with CTAC target cells were 5.5-6.5 microns in diameter, and were characterized by a reniform nucleus and varying numbers of electron-dense cytoplasmic granules.