Rice grassy stunt virus (RGSV), a member of the genus Tenuivirus, is composed of a single nucleocapsid protein and six genomic ssRNA segments. Based on the known RNA sequence of the RGSV-IR isolate, primers were designed and cDNA of the NS3 gene was obtained by reverse transcription of the virus sense (v) RNA3 and PCR amplification, with genomic RNAs of RGSV-SX isolate as template. The cDNA was then cloned and sequenced. The NS3 gene comprised 588 nt and the sequence identities were 99.1%, 96.2% at the nucleotide level and 98.4%, 96.4% at the amino acid level, compared with those of RGSV-IR and -SC isolates. The 22.9 kDa NS3 protein encoded by RGSV-SX vRNA3 showed 33.0% identity to the 21.6 kDa protein encoded by vRNA5 of RGSV over a span of 80 amino acids, and no other significant matches were found in the GenBank database. The NS3 gene was cloned in pGEX-2T to generate a prokaryotic expression plasmid pGTNS3, and a fusion protein of GST-NS3 at 49.0 kDa was induced in Escherichia coli. Antiserum prepared to this protein reacted with NS3 encoded by RGSV in Western blots. The protein was detected only in infected rice (Oryza sativa), not in purified virus or viruliferous planthopper vectors. Thus NS3 appears to be a protein encoded abundantly only while the virus infects the rice host plant and may be related to the virus pathogenesis.