DNA strand and enzyme subunit specificities involved in the interaction between E. coli RNA polymerase and T7 DNA were studied by photo-crosslinking techniques. In non-specific enzyme-DNA complexes, subunits σ, β, and β′ were crosslinked to both strands of the DNA. Under conditions leading to specific enzyme-promoter complexes, however, only σ and β subunits were crosslinked. The σ subunit was crosslinked preferentially to the non-sense strand at promoter sites. No such strand specificity was observed for the β subunit. These results provide insight into the molecular mechanism of promoter recognition and indicate that the interaction between RNA polymerase and DNA template is different at promoters and at non-specific sites.