The Ewing Sarcoma (EWS) protein is a ubiquitously expressed RNA processinhg factor that localises predominantly to the nucleus. However, the mechanism through which EWS enters the nucleus remains unclear, with differing reports identifying three separate import signals within the EWS protein. Here we have utilized a panel of truncated EWS proteins to clarify the reported nuclear localisation signals. We describe three c-terminal domains that are important for efficient EWS nuclear localization: 1) the third RGG-motif; 2) the last 10 amino acids (known as the PY-import motif); and 3) the zinc-finger motif Although these three domains are involved in nuclear import, they are not independently capable of driving the efficient import of a GFP-moiety. However, collectively they form a complex tripartite signal that efficiently drives GFP-import into the nucleus. This study helps clarify the EWS import signal, and the identification of the involvement of both the RGG- and zinc finger motifs has wide reaching implications. (C) 2009 Elsevier Inc. All rights reserved.