AbstractThis unit describes DNA‐dependent, RNA‐dependent, and template‐independent RNA polymerases. DNA‐dependent RNA polymerases include the related bacteriophage T7, T3, and SP6 polymerases, the most commonly used RNA polymerases for in vitro transcription reactions. Reaction conditions to produce preparative quantities of transcribed RNA and labeled RNA probes are covered, as are the major applications of these reactions. Limitations of the E. coli RNA polymerase for these applications are also presented. The properties of the phi6 RNA‐dependent RNA polymerase (RdRp) and its use in RNAi experiments are also introduced. Poly(A) polymerase, a template‐independent polymerase, catalyzes the incorporation of AMP residues onto the free 3′‐hydroxyl terminus of RNA, utilizing ATP as a precursor. Specific reaction conditions of poly(A) polymerase, as well as applications including RNA tailing and 3′ end labeling, are discussed. Curr. Protoc. Mol. Biol. 84:3.8.1‐3.8.8. © 2008 by John Wiley & Sons, Inc.