Cytotoxic killing assays represent an important experimental tool in immunology to determine specific and/or cross-killing activities of immune effector cells in vitro. To evaluate the killing capacity of effector cells using cytotoxic killing assays, target cells are prelabeled with a radioisotope. Present labeling methods, using 51chromium to prelabel target cells, are associated with some disadvantages. In this study, we tested the use of 35sulfur-methionine ([35S]methionine) to prelabel target cells in long term cytotoxic killing assays. Our results indicate that prelabeling of target cells with [35S]methionine allows the use of a smaller amount of target and effector cells and enables more accurate detection of target cell killing, compared to the presently used labeling methods.