To extend the nascent transcript, RNA polymerases must melt the DNA duplex downstream from the active site to expose the next acceptor base for substrate binding and incorporation. A number of mechanisms have been proposed to account for the manner in which the correct substrate is selected, and these differ in their predictions as to how far the downstream DNA is melted. Using fluorescence quenching experiments, we provide evidence that cellular RNA polymerases from bacteria and yeast melt only one DNA base pair downstream from the active site. These data argue against a model in which multiple NTPs are lined up downstream of the active site.