MLN64 is composed of two conserved regions. The amino terminal contains a conserved membrane spanning MENTAL domain shared with an unique protein called MENTHO, and targets the protein to late endosome. The carboxy-terminal domain is composed of a cholesterol binding START domain exposed to the cytoplasm. MENTHO overexpression leads to the accumulation of enlarged endosomes. In the present study we show that MLN64 overexpression also induces the formation of enlarged endosomes, an effect likely mediated by the MENTAL domain. Using an in vivo photocholesterol binding assay, we find that the MENTAL domain of MLN64 is a cholesterol binding domain. Moreover, GST pull-down or co-immunoprecipitation experiments demonstrate that this domain mediates homo and hetero-interaction of MLN64 and MENTHO. In living cells, expression of paired YFP and CFP fusion proteins show MENTHO homo-interaction and its interaction with MLN64. These data indicate that within late endosomes membranes, MLN64 and MENTHO define discrete cholesterol-containing subdomains. The MENTAL domain might serve to maintain cholesterol at the membrane of late endosomes prior to its shuttle to cytoplasmic acceptor(s).