The substrate binding site on E. coli RNA polymerase was investigated by photoaffinity labeling with a photoaffinity analog of UTP, 5-azido-UTP. We have established that 5-azido-UTP is a substrate for RNA polymerase by specific transcription on 229 bp DNA containing the gene II promoter of M13 phage. Analysis of the initial rate of RNA synthesis gives Km(5-azido-UTP) approximately 80 microM. Photolabeling with varying concentrations of 5-azido-UTP follows a saturation curve with the midpoint occurring at a 5-azido-UTP concentration of 65 microM near to the Km obtained by kinetic analysis. 5-Azido-UTP photolabels the beta', beta, and sigma subunits to about the same extent, both in the presence (33, 31, and 36%) and absence (35, 30 and 35%) of DNA. This labeling pattern is somewhat different from that obtained with 8-azido-ATP (beta' greater than sigma much greater than beta greater than alpha).