The Bell protein of human foamy virus is a 300-amino-acid nuclear regulatory protein which transactivates the gene expression directed by the homologous long terminal repeat and the human immunodeficiency virus type 1 long terminal repeat. While previous reports suggested that the single basic domain of Bell from residues 211 to 222 and/or 209 to 226 is necessary and sufficient for efficient nuclear localization (L. K. Venkatesh, C. Yang, P. A. Theodorakis, and G. Chinnandurai, J. Virol. 67:161-169, 1993; F. He, J. D. Sun, E. D. Garrett, and B. R. Cullen, J. Virol. 67:1896-1904, 1993), our recent data showed that another basic domain, from amino acid residues 199 to 200, is also required for nuclear localization of Bell (C. W. Lee, C. Jun, K. J. Lee, and Y. C. Sung, J. Virol. 68:2708-2719, 1994). To clarify this discrepancy, we constructed various bell-lacZ chimeric constructs and several linker insertion mutants and determined their subcellular localization. When the region of Bell containing basic domains was placed at an internal site of the lacZ gene, the nuclear localization signal (NLS) of Bell consisted of two discontinuous basic regions separated by an intervening sequence. Moreover, insertion of specific amino acids between two basic regions disrupted the activity of the Bell NLS. On the other hand, Bell residues 199 and 200 were not required to direct the Bell-β-galactosidase chimeric protein to the nucleus when the Bell NLS was appended to the amino terminus of β- galactosidase. These results indicate that the function of the Bell NLS is sensitive to the protein context within which the sequence is present. In addition, we demonstrated that the Bell protein forms a multimeric complex in the nuclei of mammalian cells by using a sensitive in vivo protein-protein interaction assay. Mutational analyses revealed that the regions which mediate multimer formation map to three domains of Bell, i.e., residues 1 to 31, 42 to 82, and 82 to 111. Furthermore, our results show that the region of Bell from residues 202 to 226 prevents Bell from forming a multimeric complex.