Abstract: Yeast cells contain two Bro1-domain proteins: Bro1, which is required for endosomal trafficking, and Rim20, which is required for the response to external pH via the Rim101 pathway. Rim20 associates with endosomal structures under alkaline growth conditions, when it promotes activation of Rim101 through proteolytic cleavage. We report here that the pH-dependent localization of Rim20 is contingent on the amount of Bro1 in the cell. Cells that lack Bro1 have increased endosomal Rim20-GFP under acidic conditions; cells that over-express Bro1 have reduced endosomal Rim20-GFP under acidic or alkaline conditions. The novel endosomal association of Rim20-GFP in the absence of Bro1 requires ESCRT components including Vps27, but not specific Rim101 pathway components such as Dfg16. Vps27 influences localization of Bro1 but is not required for activation of RIM101 pathway in wild-type cells, thus suggesting that Rim20 enters the Bro1 localization pathway when a vacancy exists. Despite altered localization of Rim20, the lack of Bro1 does not bypass the need for signaling protein Dfg16 to activate Rim101, as evidenced by expression levels of the Rim101 target genes RIM8 and SMP1. Therefore, endosomal association of Rim20 is not sufficient to promote Rim101 activation.