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The heat shock protein HSP90, which is mainly cytoplasmic, has recently been reported to be present in the nucleus. We have found a specific chromosomal localization of HSP90 in different species of Drosophila and Chironomus using immunocytochemical techniques with different mono- and polyclonal antibodies for this hsp. HSP90 was found associated with heat shock-induced puffs at 93D and 48B in salivary gland chromosomes of Drosophila melanogaster and Drosophila hydei, respectively. The localization of HSP90 to locus 93D occurred rapidly after the onset of heat shock and disappeared during recovery, concomitant with puff regression. The association of HSP90 with the 93D locus was strictly heat shock dependent as shown by the absence of HSP90 in puff 93D induced by either benzamide or colchicine. No specific nuclear staining was observed in unstressed control cells. HSP90 was also found in the temperature-induced telomeric Balbiani ring puffs (T-BRs) in Chironomus thummi and in one heat shock puff at I-1C in Chironomus tentans. Other heat shock puffs also appeared lightly stained with the HSP90 polyclonal antibody in both species of Chironomus. HSP90 was absent from the T-BRs when RNA synthesis was inhibited with Actinomycin D suggesting that the localization of HSP90 is dependent on transcription. Inhibition of protein synthesis did not prevent association of this hsp with the T-BRs, indicating that pre-existing HSP90 can associate with this locus. HSP90 did not associate with any telomeric chromosomal regions of unstressed cells. The present observations suggest that heat shock gene products such as HSP90 may somehow be involved in the regulation at the chromosomal level of other members of the heat shock gene family. Puffs 93D (D. melanogaster) and 48B (D. hydei) are equivalent and correspond to homologous gene loci (hsr omega) that have unusual features that distinguish them from other heat shock puffs. The binding of HSP90 at T-BRs and at puff I-1C in the genus Chironomus is the first demonstration, albeit indirect, of the existence of hsr omega analogous loci in species other than Drosophila.