Thermal variations can exert dramatic effects on the rates of enzymes. The influence of temperature on RNA polymerase is of particular interest because its transcriptional activity governs general levels of gene expression, and may therefore exhibit pleiotropic effects in cells. Using a custom-modified optical trapping apparatus, we used a tightly focused infrared laser to heat single molecules of Escherichia coli RNA polymerase while monitoring transcriptional activity. We found a significant change in rates of transcript elongation with temperature, consistent with a large enthalpic barrier to the condensation reaction associated with RNA polymerization (approximately 13 kcal/mol). In contrast, we found little change in either the frequency or the lifetime of off-pathway, paused states, indicating that the energetic barrier to transcriptional pausing is predominantly entropic.