AR9 is a giant Bacillus subtilis phage whose uracilcontaining double-stranded DNA genome encodes distant homologs of β and β' subunits of bacterial RNA polymerase (RNAP). The products of these genes are thought to assemble into two noncanonical multisubunit RNAPs - a virion RNAP (vRNAP) that is injected into the host along with phage DNA to transcribe early phage genes, and a nonvirion RNAP (nvRNAP), which is synthesized during the infection and transcribes late phage genes. We purified the AR9 nvRNAP from infected B. subtilis cells and characterized its transcription activity in vitro. The AR9 nvRNAP requires uracils rather than thymines at specific conserved positions of late viral promoters. Uniquely, the nvRNAP recognizes the template strand of its promoters and is capable of specific initiation of transcription from both doubleand single-stranded DNA. While the AR9 nvRNAP does not contain homologs of bacterial RNAP β subunits, it contains, in addition to the β and β'-like subunits, a phage protein gp226. The AR9 nvRNAP lacking gp226 is catalytically active but unable to bind to promoter DNA. Thus, gp226 is required for promoter recognition by the AR9 nvRNAP and may represent a new group of transcription initiation factors.