Efficient antitumor immune response requires the coordinated function of integrated immune components, but is finally exerted by the differentiated effector tumor-infiltrating lymphocytes (TIL). TIL cells comprise, therefore, an exciting platform for adoptive cell transfer (ACT) in cancer. In this study, we show that the inhibitory carcinoembryonic Ag cell adhesion molecule 1 (CEACAM1) protein is found on virtually all human TIL cells following preparation protocols of ACT treatment for melanoma. We further demonstrate that the CEACAM1 homophilic interactions inhibit the TIL effector functions, such as specific killing and IFN-gamma release. These results suggest that CEACAM1 may impair in vivo the antitumor response of the differentiated TIL. Importantly, CEACAM1 is commonly expressed by melanoma and its presence is associated with poor prognosis. Remarkably, the prolonged coincubation of reactive TIL cells with their melanoma targets results in increased functional CEACAM1 expression by the surviving tumor cells. This mechanism might be used by melanoma cells in vivo to evade ongoing destruction by tumor-reactive lymphocytes. Finally, CEACAM1-mediated inhibition may hinder in many cases the efficacy of TIL ACT treatment of melanoma. We show that the intensity of CEACAM1 expression on TIL cells constantly increases during ex vivo expansion. The implications of CEACAM1-mediated inhibition of TIL cells on the optimization of current ACT protocols and on the development of future immunotherapeutic modalities are discussed.