MAGE-A genes are expressed by a variety of cancers but not by normal adult tissues, except for testis and placenta. They encode 12 highly homologous proteins of 309-369 aminoacids (De Plaen et al., 1994). Several peptide epitopes from MAGE-A proteins, including MAGE-1, 3, 6 and 10, recognized by specific cytolytic T lymphocytes derived from tumors or peripheral blood have been described (Van den Eynde and van der Bruggen, 1997; Huang et al., 1999; Zorn and Hercend, 1999). In addition, HLA class II restricted epitopes have been described for MAGE-3 (Chaux et al., 1999; Manici et al., 1999). MAGE-A genes are therefore regarded as attractive candidates for specific immunotherapy and clinical trials of cancer patients involving immunization with MAGE-A1 and 3 peptides are in progress (Marchand et al., 1999; Nestle et al., 1998). Monoclonal antibodies (MAbs) against MAGE-1, 3, 4 and 11 proteins have been developed using the respective recombinant E. coli proteins as immunogen (Chen et al., 1994; Kocher et al., 1995; Shichijo et al., 1995; Carrel et al., 1996; Jurk et al., 1998). The anti-MAGE-3 MAb 57B appeared to be particularly interesting as it was shown to be suitable for immunocytochemistry for both frozen and paraffin-embedded tumor samples (Hofbauer et al., 1997). However, the possibility that crossreactivity of this antibody with other members of the MAGE-A family may exist, due to the high degree of homology of these proteins (MAGE-3 and 6, in particular, differ only for 11 aminoacids), has not been formally ruled out. To this regard, we have demonstrated that a MAb raised against recombinant MAGE-1, named 6C1, strongly cross-reacts with MAGE-10 (Carrel et al., 1996; Rimoldi et al., 1999). To resolve this issue, we have investigated in detail the cross-reactivities of anti-MAGE-3 MAb 57B and anti-MAGE-1 MAb 6C1 using a transient transfection approach where human embryonic kidney 293T cells were used as recipient. Plasmids containing cDNAs encoding the more commonly expressed MAGE genes (MAGE-1, 2, 3, 4, 6, 10 and 12) were transfected individually and cell lysates analyzed by Western blotting. Figure 1a shows that both MAbs were able to recognize all the different MAGE proteins, except for MAGE-10 that was not recognized by MAb 57B. These results demonstrate the high degree of cross-reactivity of both MAbs and also show that all MAGE-A proteins have an apparent mw of 45–50 kDa, except for MAGE-10, which displays an unexpected MW of 72 kDa (Rimoldi et al., 1999). The extent of cross-reactivity towards the different proteins appeared to be variable. However, quantitative interpretations cannot be drawn from these experiments due to possible variations in transfection efficiencies. To confirm that cross-reactivity by the MAbs can take place when MAGE genes are expressed at a normal physio logical level, such as found in tumors, a series of cell lines expressing different combinations of MAGE genes, as determined by RT/PCR, was analyzed. Figure 1b confirms the ability of the 2 MAbs to recognize multiple members of the MAGE family. In particular, MAb 57B detected a band of approximately 50 kDa (similar to the size of MAGE-3, Kocher et al., 1995) in melanoma Me242 and Me244 cells (expressing only