A photoactive nucleotide analogue of UTP, 5-azido-uridine 5'-triphosphate (5-N3UTP), has been demonstrated to interact with the RNA polymerase of the vesicular stomatitis virus (VSV) transcription complex. Kinetic studies indicated that 5-N3UTP served as an efficient replacement for UTP in in vitro polymerase reactions. The Km for the azido analogue was 27 microM and that of the natural substrate, UTP, was 7 microM. Photolysis of [gamma-32P]5-N3UTP in the presence of VSV transcription complexes resulted in selective radio-labelling of the L protein. This photolabelling was saturable with an apparent Kd of 28 microM. The L protein was protected from [gamma-32P]5-N3UTP-mediated photolabelling by competing natural substrates (UTP, CTP, ATP, GTP). The stoichiometry of photoprobe incorporation into the transcription complex was close to unity with respect to the L protein. These data provide evidence that the nucleotide-binding domain of the VSV RNA polymerase contains amino acid residues of the L protein.