Fluorescence RNA:RNA in situ hybridization studies in various larval and adult cell types of Drosophila melanogaster showed that the noncoding hsr-omega nuclear (hsromega-n) transcripts were present in the form of many small speckles. These speckles, which we name 'omega speckles', were distributed in the interchromatin space in close proximity to the chromatin. The only chromosomal site where hsromega-n transcripts localized was the 93D locus or the hsromega gene itself. The number of nucleoplasmic speckles varied in different cell types. Heat shock, which inhibits general chromosomal transcription, caused the individual speckles to coalesce into larger but fewer clusters. In extreme cases, only a single large cluster of hsromega-n transcripts localizing to the hsromega locus was seen in each nucleus. In situ immunocytochemical staining using antibodies against heterogenous nuclear RNA binding proteins (hnRNPs) like HRB87F, Hrp40, Hrb57A and S5 revealed that, in all cell types, all the hnRNPs gave a diffuse staining of chromatin areas and in addition, were present as large numbers of speckles. Colocalization studies revealed an absolute colocalization of the hnRNPs and the omegaspeckles. Heat shock caused all the hnRNPs to cluster together exactly, following the hsromega-n transcripts. Immunoprecipitation studies using the hnRNP antibodies further demonstrated a physical association of hnRNPs and hsromega transcripts. The omegaspeckles are distinct from interchromatin granules since nuclear speckles containing serine/arginine-rich SR-proteins like SC35 and SRp55 did not colocalize with the &ohgr; speckles. The speckled distribution of hnRNPs was completely disrupted in hsromega nullosomics. We conclude that the hsromega-n transcripts play essential structural and functional roles in organizing and establishing the hnRNP-containing omega speckles and thus regulate the trafficking and availability of hnRNPs and other related RNA binding proteins in the cell nucleus.