At 739 amino acids, the nucleoprotein (NP) of Ebola virus is the largest nucleoprotein of the nonsegmented negative-stranded RNA viruses, and like the NPs of other viruses, it plays a central role in virus replication. Huang et al. (Y. Huang, L. Xu, Y. Sun, and G. J. Nabel, Mol. Cell 10:307-316, 2002) previously demonstrated that NP, together with the minor matrix protein VP24 and polymerase cofactor VP35, is necessary and sufficient for the formation of nucleocapsid-like structures that are morphologically indistinguishable from those seen in Ebola virus-infected cells. They further showed that NP is O glycosylated and sialylated and that these modifications are important for interaction between NP and VP35. However, little is known about the structure-function relationship of Ebola virus NP. Here, we examined the glycosylation of Ebola virus NP and further investigated its properties by generating deletion mutants to define the region(s) involved in NP-NP interaction (self-assembly), in the formation of nucleocapsid-like structures, and in the replication of the viral genome. We were unable to identify the types of glycosylation and sialylation, although we did confirm that Ebola virus NP was glycosylated. We also determined that the region from amino acids 1 to 450 is important for NP-NP interaction (self-assembly). We further demonstrated that these amino-terminal 450 residues and the following 150 residues are required for the formation of nucleocapsid-like structures and for viral genome replication. These data advance our understanding of the functional region(s) of Ebola virus NP, which in turn should improve our knowledge of the Ebola virus life cycle and its extreme pathogenicity.