The present work was designed to compare two mechanisms of cellular recognition based on Ab specificity: firstly, when the anti-HER2 mAb trastuzumab bridges target cells and cytotoxic lymphocytes armed with a Fc receptor (ADCC) and, secondly, when HER2 positive target cells are directly recognized by cytotoxic lymphocytes armed with a chimeric antigen receptor (CAR). To compare these two mechanisms, we used the same cellular effector (NK-92) and the same signaling domain (Fcí µí¼€RIí µí»¾). The NK-92 cytotoxic cell line was transfected with either a Fcí µí»¾RIIIa-Fcí µí¼€RIí µí»¾ (NK-92 CD16) or a trastuzumab-based scFv-Fcí µí¼€RIí µí»¾ chimeric receptor (NK-92 CAR). In vitro, the cytotoxic activity against HER2 positive target cells after indirect recognition by NK-92 CD16 was always inferior to that observed after direct recognition by NK-92 CAR. In contrast, and somehow unexpectedly, in vivo, adoptive transfer of NK-92 CD16 + trastuzumab but not of NK-92 CAR induced tumor regression. Analysis of the in vivo xenogeneic system suggested that the human CH2-CH3 IgG2 used as a spacer in our construct was able to interact with the FcR present at the cell surface of the few NSG-FcR+ remaining immune cells. This interaction, leading to blockage of the NK-92 CAR in the periphery of the engrafted tumor cells, stresses the critical role of the composition of the spacer domain.