In vivo UV cross-linking and nuclear transcriptional run-on experiments have shown that a number of Drosophila genes possess an elongationally paused RNA polymerase on their 5' ends. Here, we examine in vivo promoters that do and do not possess paused polymerases using the single-stranded DNA-probing reagent KMnO4. Melted DNA helices are found associated with the pause site of the uninduced hsp70 and hsp26 heat shock genes and the constitutively expressed beta-1 tubulin gene. The histone H1 and H2B genes, which lack a paused polymerase, have no comparable region of melted DNA. Melting at the pause site persists upon heat shock induction of the hsp70 and hsp26 genes, indicating that pausing continues after gene activation. Interestingly, activation triggers additional melting, both at the start site (in the region where open complexes would be expected to form) and downstream of the uninduced pause site. In the course of our studies, we discovered that some T residues of the TATA box were protected from KMnO4 modification in both induced and uninduced cells. This protection appears to be a consequence of TFIID binding, as a similar protection pattern could be produced in vitro with purified protein.