Using an interaction blot approach to search in the human nuclear proteome, we identified eight novel proteins that bind the hyperphosphorylated C-terminal repeat domain (phosphoCTD) of RNA polymerase II. Unexpectedly, five of the new phosphoCTD-associating proteins (PCAPs) represent either enzymes that act on DNA and chromatin (topoisomerase I, DNA (cytosine-5) methyltransferase 1, poly(ADP-ribose) polymerase-1) or proteins known to bind DNA (heterogeneous nuclear ribonucleoprotein (hnRNP) U/SAF-A, hnRNP D). The other three PCAPs represent factors involved in pre-mRNA metabolism as anticipated (CA150, NSAP1/hnRNP Q, hnRNP R) (note that hnRNP U/SAF-A and hnRNP D are also implicated in pre-mRNA metabolism). Identifying as PCAPs proteins involved in diverse DNA transactions suggests that the range of phosphoCTD functions extends far beyond just transcription and RNA processing. In view of the activities possessed by the DNA-directed PCAPs, it is likely that the phosphoCTD plays important roles in genome integrity, epigenetic regulation, and potentially nuclear structure. We present a model in which the phosphoCTD association of the PCAPs poises them to act either on the nascent transcript or on the DNA/chromatin template. We propose that the phosphoCTD of elongating RNA polymerase II is a major organizer of nuclear functions.