To improve radioimmunotherapy with Auger electron emitters, we assessed whether the biological efficiency of (125)I varied according to its localization. A-431 and SK-OV-3 carcinoma cells were incubated with increasing activities (0-4 MBq/ml) of (125)I-labeled vectors targeting the cell membrane, the cytoplasm or the nucleus. We then measured cell survival by clonogenic assay and the mean radiation dose to the nucleus by assessing the cellular medical internal radiation dose (MIRD). The relationship between survival and the radiation dose delivered was investigated with a linear mixed regression model. For each cell line, we obtained dose-response curves for the three targets and the reference values (i.e., the dose leading to 75, 50 or 37% survival). When cell survival was expressed as a function of the total cumulative decays, nuclear (125)I disintegrations were more harmful than disintegrations in the cytoplasm or at the cell membrane. However, when survival was expressed as a function of the mean radiation dose to the nucleus, toxicity was significantly higher when (125)I was targeted to the cell membrane than to the cytoplasm. These findings indicate that the membrane is a more sensitive target than the cytoplasm for the dense ionization produced by Auger electrons. Moreover, cell membrane targeting is as cytotoxic as nuclear targeting in SK-OV-3 cells. We suggest that targeting the membrane rather than the cytoplasm may contribute to the development of more efficient radioimmunotherapies based on Auger electron radiation, also because most of the available vectors are directed against cell surface antigens.