The nucleocapsid (N) protein of hantaviruses encapsidates both viral genomic and antigenomic RNAs, although only the genomic viral RNA (vRNA) is packaged into virions. To define the domain within the Hantaan virus (HTNV) N protein that mediates these interactions, 14 N- and C-terminal deletion constructs were cloned into a bacterial expression vector, expressed, and purified to homogeneity. Each protein was examined for its ability to bind the HTNV S segment vRNA with filter binding and gel electrophoretic mobility shift assays. These studies mapped a minimal region within the HTNV N protein (amino acids 175 to 217) that bound vRNA. Sequence alignments made from several hantavirus N protein sequences showed that the region identified has a 58% identity and an 86% similarity among these amino acid sequences. Two peptides corresponding to amino acids 175 to 196 (N1) and 197 to 218 (N2) were synthesized. The RNA binding of each peptide was measured by filter binding and competition analysis. Three oligoribonucleotides were used to measure binding affinity and assess specificity. The N2 peptide contained the major RNA binding determinants, while the N1 peptide, when mixed with N2, contributed to the specificity of vRNA recognition.