A conserved ambient pH signal transduction pathway has been evidenced in both ascomycetous yeasts and filamentous fungi, called the Rim or Pal pathway, respectively. However, closely related PalC orthologues are found only in Yarrowia lipolytica and in filamentous fungi, where the Rim9p/PalI factor has a much longer C-terminal tail than in other yeasts. We show here that, like Aspergillus nidulans palI mutants, a Ylrim9Delta mutant has a less extreme phenotype than other mutants of the pathway, whereas rim9 mutants in Saccharomyces cerevisiae and Candida albicans reportedly exhibit a tight Rim phenotype. Deletion of the long C-terminal tail of YlRim9p/PalI had no phenotypic effect on ambient pH signalling. We also show that the Y. lipolytica PalC orthologue, named YlRim23p, is absolutely required for the alkaline pH response. Its only interactant identified in a genome-wide two-hybrid screen is YlSnf7/Vps32p, confirming the link between the Rim and the Vps pathways. YlRim13p and YlRim20p both interact with YlSnf7/Vps32p but not with YlRim23p. The long C-terminal tail of YlRim9p/PalI interacts neither with YlRim23p nor with YlSnf7/Vps32p. These results show that YlRim23p is a bona fide component of the Rim pathway in Y. lipolytica and that it participates in the complexes linking pH signalling and endocytosis.