Huntingtin-associated protein 1 (HAP1) is a novel protein of unknown function with a higher binding affinity for the mutant form of Huntington's disease protein huntingtin. Here we report that HAP1 interacts with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a mammalian homologue of yeast vacuolar protein sorting protein Vps27p involved in the endosome-to-lysosome trafficking. This novel interaction was identified in a yeast two-hybrid screen using full-length Hrs as bait, and confirmed by in vitro binding assays and co-immunoprecipitation experiments. Deletion analysis reveals that the association of HAP1 with Hrs is mediated via a coiled-coil interaction between the central coiled-coil domains of both proteins. Immunofluorescence and subcellular fractionation studies show that HAP1 co-localizes with Hrs on early endosomes. Like Hrs, overexpression of HAP1 causes the formation of enlarged early endosomes, and inhibits the degradation of internalized epidermal growth factor receptors. Whereas overexpression of HAP1 does not affect either constitutive or ligand-induced receptor-mediated endocytosis, it potently blocks the trafficking of endocytosed epidermal growth factor receptors from early endosomes to late endosomes. These findings implicate, for the first time, the involvement of HAP1 in the regulation of vesicular trafficking from early endosomes to the late endocytic compartments.