JIL-1 is the major kinase controlling the phosphorylation state of histone H3S10 at interphase in Drosophila. In this study, we used three different commercially available histone H3S10 phosphorylation antibodies, as well as an acid-free polytene chromosome squash protocol that preserves the antigenicity of the histone H3S10 phospho-epitope, to examine the role of histone H3S10 phosphorylation in transcription under both heat shock and non-heat shock conditions. We show that there is no redistribution or upregulation of JIL-1 or histone H3S10 phosphorylation at transcriptionally active puffs in such polytene squash preparations after heat shock treatment. Furthermore, we provide evidence that heat shock-induced puffs in JIL-1 null mutant backgrounds are strongly labeled by antibody to the elongating form of RNA polymerase II (Pol IIoser2), indicating that Pol IIoser2 is actively involved in heat shock-induced transcription in the absence of histone H3S10 phosphorylation. This is supported by the finding that there is no change in the levels of Pol IIoser2 in JIL-1 null mutant backgrounds compared with wild type. mRNA from the six genes that encode the major heat shock protein in Drosophila, Hsp70, is transcribed at robust levels in JIL-1 null mutants, as directly demonstrated by qRT-PCR. Taken together, these data are inconsistent with the model that Pol II-dependent transcription at active loci requires JIL-1-mediated histone H3S10 phosphorylation, and instead support a model in which transcriptional defects in the absence of histone H3S10 phosphorylation are a result of structural alterations of chromatin.