Puffs in the polytene chromosome of Drosophila melanogaster are characteristic of sites of high-level active transcription which can be observed directly under the microscope. We studied the dependence of puff formation on chromatin modifications at a site where a GAL4-inducible transgene is located in the 61C7 cytological region. Immunostaining of salivary gland polytene chromosomes indicated no increase of either dSAGA-specific histone H3 lysine 14, or ATAC-specific histone H4 lysine 5 and 12 acetylation in the puffed region. Nor did we observe increased levels of H4K8ac, H3K18ac, or H4K16ac in the puff. In accordance with the above, puff formation as well as localization of Pol II and GAL4 was detectable at the 61C region in dAda2b and dAda2a null homozygotes, which are dSAGA- and ATAC-specific mutants, respectively. Moreover, the reduced level of JIL-1-specific H3 serine 10 phosphorylation did not abolish puff formation in ATAC mutants. Surprisingly, in wild-type animals dADA3 and GCN5 shared constituents of dSAGA and ATAC, as well as JIL-1 localized specifically to the puff, where the JIL-1-phosphorylated H3S10ph level was also high. Altogether these data strongly suggest that the GAL4 activator can induce transcription and chromatin reorganization seen as a puff without dSAGA- and ATAC-specific histone acetylation and JIL-1-specific histone H3 phosphorylation.