In this study, we have measured the specific tumoricidal activity of tumor-infiltrating lymphocytes that prevent the growth of secondary tumors in animals harboring progressing primary tumors (concomitant immunity). Since no tumor grows at the challenge site when concomitant immunity is established, tumor cells were inoculated into a preimplanted gelatin sponge whose subsequent solubilization in collagenase permitted the retrieval of leukocytes after tumor challenge. Primary progressing EMT6 tumors were established in normal BALB/c mice and 10 days later they were challenged with a secondary tumor inoculum introduced through a preimplanted gelatin sponge. At 3, 7, and 10 days after the administration of the tumor inoculum challenge, a monodispersed suspension of infiltrating leukocytes was recovered by collagenase digestion of the sponge matrix and tested for cytotoxicity toward EMT6 tumor targets. Cytotoxic T-lymphocytes with tumoricidal activity accumulated at the site of the secondary tumor challenge by 3 days. This antitumor activity was maximal 7 days following challenge and decayed thereafter. Splenic lymphocytes from these animals showed little cytotoxicity. In animals harboring a primary tumor, lymphocytes found in sponges that were not inoculated with tumor cells were not cytotoxic. We interpret these data to indicate that cytotoxic lymphocytes migrate to, and accumulate at the site of the tumor but not at other sites and that peripheral sources of lymphocytes in tumor-bearing animals such as the spleen may not be the best source of effector cells for evaluating the host's immune response to its tumor. The approach described here may also be useful in studying the mechanisms for host control of metastatic disease.