Sin Nombre (SN) virus is the major etiologic agent of hantavirus pulmonary syndrome, a severe respiratory disease with high mortality. Like other hantaviruses, SN virus causes an inapparent chronic infection of the natural rodent reservoir and tends to grow slowly and produce little cytopathic effect even in highly susceptible Vero E6 tissue culture cells. An electrochemiluminescent quantitative PCR approach was developed to allow examination of SN virus RNA transcription in synchronously infected cells. Although virion particles contain equimolar ratios of the three negative-strand genome RNA segments (S, M, and L), rates and levels of accumulation of the corresponding N, GPC, and L viral mRNAs varied. The smallest mRNA (N) was detectable earliest and plateaued at the highest level, where as the largest mRNA (L) appeared latest and at the lowest plateau level. In addition, all three mRNAs were found to share a common 5' capped primer initiation mechanism, but appeared to have different mRNA termination mechanisms. The N mRNA 3' terminus mapped to position 1435 on the S segment, in close proximity to a CCC-rich suspected transcription termination motif. The GPC mRNA 3' terminus contained a poly(A) tall and mapped to a U8 transcription termination-polyadenylation motif reminiscent of those seen in other negative-strand RNA viruses. Finally, the L mRNA 3' terminus appeared identical to the L segment antigenome 3' terminus.