The genes belonging to the B-box family have been implicated in a variety of processes. Among them, PML, RFP, and Tif1 acquire oncogenic activity when fused to RAR alpha, RET, and B-raf, respectively. Recently, two additional B-box genes involved in human diseases have been identified, the FMF gene which is mutated in Familial Mediterranean Fever, and the MID1 gene, involved in X-linked Opitz syndrome. These genes contain a RING, two B-boxes, a coiled-coil, and a frequently conserved C-terminal domain. Very little is known about the molecular mechanisms mediating the function of B-box genes and thus we decided to redefine and characterize the entire family using a systematic and comprehensive approach to efficiently move from sequence/structure information to functional knowledge. We identified novel members of the B-box family by a systematic search of the dbEST and we have collected the full-length cDNA of the 15 known and 18 novel B-box genes. To evaluate the possible involvement of the B-box genes in human diseases, we have defined their chromosomal position and analyzed their expression patterns in adult tissues and during development. In addition, we have designed functional assays which allowed us to find that many family members show a strong growth suppression potential and are able to repress transcription. Moreover, we found that most of the B-box proteins are able to homodimerize and to form large multiprotein complexes. The B-box proteins, through their ability to form multiprotein complexes, appear to play an extremely important role in defining different subcellular compartments, both in the nucleus and in the cytoplasm, suggesting a possible involvement in compartimentalizing other proteins. These data will be useful to better understand the role of this class of proteins during normal development and to assess their role in the pathogenesis of tumors and inherited diseases.