SP6 is a small, virulent bacteriophage which grows on Salmonella typhimurium LT2. It is morphologically similar to Escherichia coli bacteriophage T7 and its relatives, but appears to be genetically distinct. After infection a bacteriophage-specific RNA polymerase is induced in infected cells. SP6 RNA polymerase is a stable enzyme and is easily purified to homogeneity in good overall yield. The activity resides in a single polypeptide chain of Mr = 96,000. Synthesis of RNA by SP6 RNA polymerase requires a DNA template and Mg2+ ion and is strongly stimulated by either bovine serum albumin of spermidine. Thiol-reactive reagents inhibit the enzyme, suggesting the presence of essential sulfhydryl residues. RNA synthesis requires native SP6 RNA as template; DNAs from other bacteriophages including T3 and T7 are inert; hence, SP6 RNA polymerase possesses a stringent promoter specificity similar to, but distinct from that of the other phage RNA polymerases. The SP6 RNA polymerase is also highly active in synthesis of poly(rG) with poly(dI) . (dC) as template. This reaction is unlikely to involve promoter-like sites, but it appears to reflect a general catalytic capacity of the polymerase, since cleavage of the SP6 RNA polymerase with trypsin, which completely eliminates SP6-transcribing activity, has little effect on poly(rG) synthesis. Hence, it appears that the catalytic portion of the polymerase can be separated from the RNA polymerase holoenzyme.