Pre-mRNA synthesis in eukaryotic cells is preceded by the formation of a transcription initiation complex and binding of unphosphorylated RNA polymerase II (Pol II) at the promoter region of a gene. Transcription initiation and elongation are accompanied by the hyperphosphorylation of the carboxy-terminal domain (CTD) of Pol II large subunit. Recent biochemical studies provided evidence that RNA processing factors, including those required for splicing, associate with hyperphosphorylated CTDs forming "transcription factories." To directly visualize the existence of such factories, we simultaneously detected human cytomegalovirus immediate-early (IE) DNA and RNA with splicing factors and Pol II in rat 9G cells inducible for IE gene expression. Combined in situ hybridization and immunocytochemistry revealed that, after induction, both splicing factors and Pol II are present at the sites of IE mRNA synthesis and of IE mRNA processing that extend from the transcribing gene. Noninduced cells revealed no such associations. When IE mRNA-synthesizing cells were treated with a transcription inhibitor, these associations disappeared within 30 min. Our results show that the association of Pol II and splicing factors with IE DNA is dependent on its transcriptional activity and furthermore suggest that splicing factors are still associated with Pol II during active splicing.