VP40, the putative matrix protein of both Ebola and Marburg viruses, possesses a conserved proline-rich motif (PY motif) at its N terminus. We demonstrate that the VP40 protein can mediate its own release from mammalian cells, and that the PY motif is important for this self-exocytosis (budding) function. In addition, we used Western-ligand blotting to demonstrate that the PY motif of VP40 can mediate interactions with specific cellular proteins that have type I WW-domains, including the mammalian ubiquitin ligase, Nedd4. Single point mutations that disrupted the PY motif of VP40 abolished the PY/WW-domain interactions. Significantly, the full-length VP40 protein was shown to interact both physically and functionally with full-length Rsp5, a ubiquitin ligase of yeast and homolog of Nedd4. The VP40 protein was multiubiquitinated by Rsp5 in a PY-dependent manner in an in vitro ubiquitination assay. These data demonstrate that the VP40 protein of Ebola virus possesses a PY motif that is functionally similar to those described previously for Gag and M proteins of specific retroviruses and rhabdoviruses, respectively. Last, these studies imply that VP40 likely plays an important role in filovirus budding, and that budding of retroviruses, rhabdoviruses, and filoviruses may proceed via analogous mechanisms.