Subunits of RNA polymerase II that come into contact with nascent RNA transcripts have been determined by photoaffinity labeling. Transcription was carried out in a cell-free extract in the presence of 4-thio-UTP, and utilized the major late promoter of adenovirus-2 DNA. The transcript length was limited by inclusion of the chain terminator 3'-O-methyl-GTP. Transcription complexes were irradiated with near-UV light (lambda greater than 300 nm) to specifically photoactivate 4-thio-uridine. After photocross-linking, radiolabeled proteins were separated by electrophoresis, blotted onto nitrocellulose, and visualized by autoradiography. Specific photoaffinity labeling of enzyme subunits IIo and IIc was observed. In some experiments, radiolabeled IIa was also detected. Based on the level of photoaffinity labeling of subunits IIo and IIa, relative to their concentration in the transcription reaction, the transcriptional activity of RNA polymerase IIO appears to be greater than 10 times that of IIA. RNA attached to subunits IIo and IIc was estimated to be 16-40 nucleotides in length, whereas RNA attached to subunit IIa was approximately 27-40 nucleotides long. Photoaffinity labeling was sensitive to alpha-amanitin, and required DNA containing an RNA polymerase II promoter.