The ALG-2-interacting protein Alix has recently been demonstrated to associate with CHMP4b that is a human homologue of yeast Snf7p (also named Vps32p) and a member of the family of small coiled-coil proteins named CHMP implicated in playing roles in multivesicular body sorting. In addition to the previously isolated cDNAs for two CHMP4 proteins (CHMP4a and CHMP4b), we isolated a cDNA for a new member of the CHMP4 subfamily (designated CHMP4c). Northern blot analyses revealed different expression patterns of the mRNAs for the three CHMP4 isoforms in human tissues. CHMP4b messages were expressed at higher levels in all 12 tissues tested in comparison with the CHMP4a and CHMP4c transcripts, particularly in heart and skeletal muscle. The interaction with Alix was detected for each CHMP4 isoform by co-immunoprecipitation experiments using lysates of HEK293 cells expressing each epitope-tagged CHMP4 protein and Alix fused with green fluorescent protein. Further, using recombinant glutathione S-transferase (GST) fusion protein of truncated Alix (amino acids 1-423) and thioredoxin-tagged CHMP4 proteins, the direct interactions were detected by a GST pull-down assay, where CHMP4b showed a stronger interaction than other CHMP4 isoforms. These results suggest that CHMP4b is a major binding partner of Alix among the three CHMP4 isoforms.