We isolated and sequenced a nuclear gene and cDNA encoding a bacteriophage T7-type RNA polymerase, NsRpoT-B, from Nicotiana sylvestris. The gene, NsRpoT-B, consists of 19 exons and 18 introns and encodes a polypeptide of 1020 amino acid residues. The predicted NsRpoT-B protein shows 71% amino acid identity with NsRpoT-A which is a mitochondrial protein. Quantitative RT-PCR revealed that steady-state NsRpoT-B mRNA accumulation is highest in the mature leaves and lowest in the cotyledons. Transient expression assays in protoplasts from N. sylvestris leaves demonstrated that the putative N-terminal transit peptide of NsRpoT-B encodes dual targeting signals directing the protein into mitochondria and plastids. This strongly suggests that NsRpoT-B functions as an RNA polymerase transcribing genes from two different plant organelle genomes. NsRpoT-B transcripts have two potential translation initiation codons. An in vitro translation assay indicated that a chimeric mRNA encoding the N-terminal NsRpoT-B fused to an sGFP produced two polypeptides translated from the first and second initiation codons. This implies that the dual targeting of NsRpoT-B protein is regulated, in part, at the level of translation. We have designated this protein NsRpoTpm.