Actin is abundant in the nucleus and has been implicated in transcription; however, the nature of this involvement has not been established. Here we demonstrate that beta-actin is critically involved in transcription because antibodies directed against beta-actin, but not muscle actin, inhibited transcription in vivo and in vitro. Chromatin immunoprecipitation assays demonstrated the recruitment of actin to the promoter region of the interferon-gamma-inducible MHC2TA gene as well as the interferon-alpha-inducible G1P3 gene. Further investigation revealed that actin and RNA polymerase II co-localize in vivo and also co-purify. We employed an in vitro system with purified nuclear components to demonstrate that antibodies to beta-actin block the initiation of transcription. This assay also demonstrates that beta-actin stimulates transcription by RNA polymerase II. Finally, DNA-binding experiments established the presence of beta-actin in pre-initiation complexes and also showed that the depletion of actin prevented the formation of pre-initiation complexes. Together, these data suggest a fundamental role for actin in the initiation of transcription by RNA polymerase II.