AbstractSeveral tumor cell systems have been developed for investigating the cytotoxic activity of different substances. The Ehrlich ascytic tumor (EAT) has been studied extensively and used to increase our comprehension of immunotherapy (Yamamoto and Naraparaju. Cancer Res 57:2187–2192, 1997). In this study, we evaluated the ability of an enzymatic method to assess cytotoxic tumor activity in vitro. To prepare cytotoxic cells, lymphoid cells isolated from the lymph nodes of C57BL/6 mice were activated with interleukin‐2 (IL‐2). The cytotoxic activity to EAT cells was assessed by an enzymatic test using lactate dehydrogenase (LDH), an enzyme widely distributed in mammalian tissues which, in the presence of NAD/NADH, converts lactate to pyruvate. IL‐2 treatment of lymph node cells induced a greater percentage of lysis (75.47%) than effector cells that were not treated with IL‐2 (42.38%). This suggests that IL‐2 directly activates effector cells and not tumor cells. The results demonstrate that the LDH release‐measurement method can be utilized to assay cytotoxic cell‐mediated activity in which murine tumor cells act as the target. Levels of IL‐2 appear to have a direct correlation to cellular immunity and treatment with these substances may strengthen immune function and decrease the pace of disease development. J. Clin. Lab. Anal. 18:27–30, 2004. © 2004 Wiley‐Liss, Inc.