To investigate functional differences between RNA polymerases IIA and II0 (Pol IIA and Pol II0), with hypoand hyperphosphorylated carboxy-terminal repeat domains (CTDs), respectively, we have visualized the in vivo distributions of the differentially phosphorylated forms of Pol II on Drosophila polytene chromosomes.Using phosphorylation state-sensitive antibodies and immunofluorescence microscopy with digital imaging, we find Pol IIA and Pol II0 arrayed in markedly different, locus-and condition-specific patterns. Major ecdysone-induced puffs, for example, stain exclusively for Pol II0, indicating that hyperphosphorylated Pol II is the transcriptionally active form of the enzyme on these genes. In striking contrast, induced heat shock puffs stain strongly for both Pol IIA and Pol II0, suggesting that heat shock genes are transcribed by a mixture of hypo-and hyperphosphorylated forms of Pol II. At the insertion sites of a transposon carrying a hybrid hsp70-lacZ transgene, we observe only Pol IIA before heat shock induction, consistent with the idea that Pol II arrested on the hsp70 gene is form IIA. After a 90-sec heat shock, we detect heat shock factor (HSF) at the transposon insertion sites; and after a 5-min shock its spatial distribution on the induced transgene puffs is clearly resolved from that of Pol II. Finally, using antibodies to hnRNP proteins and splicing components, we have discerned an apparent overall correlation between the presence and processing of nascent transcripts and the presence of Pol II0.[Key Words: RNA polymerase IIA/II0; CTD phosphorylation; transcription factors; in situ localization; immunofluorescence microscopy; digital imaging] Received July 28, 1993; revised version accepted September 29, 1993.The carboxy-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is an unusual entity composed of multiple repeats of the 7-amino-acid consensus sequence YSPTSPS (for review, see Corden 1990;Young 1991). The number of repeats ranges from 26 in yeast, to 42 in Drosophila, to 52 in mammals. The CTD has been shown to carry out essential in vivo roles in yeast (Nonet et al. 1987), Drosophila (Zehring et al. 1988), and mammalian cells (Bartolomei et al. 1988), but precisely what those roles are is not well understood. Suggested roles for the CTD include interacting with transcription initiation factors, serving as a molecular "cowcatcher" to facilitate movement of polymerase on chromatin templates, providing a link between transcription and RNA processing, and localizing polymerase to specific nuclear compartments (Corden 1990 (Allison et al. 1988;Scafe et al. 1990;Peterson et al. 1991), suggesting that the CTD may be involved in receiving regulatory signals at certain promoters. In vivo and in vitro experiments suggest that these responses might be mediated through the TATA-binding protein (TBP) component of TFIID (Koleske et al. 1992;Usheva et al. 1992;Thompson et al. 1993). On the other hand, the precise nature of the CTD involvement in initiation is not known, ...