ABSTRACT Fluorescence RNA:RNA in situ hybridization studies in various larval and adult cell types of Drosophila melanogaster showed that the noncoding hsr-omega nuclear (hsrω-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 hsrω-n transcripts localized was the 93D locus or the hsrω 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 hsrω-n transcripts localizing to the hsrω 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 omega speckles. Heat shock caused all the hnRNPs to cluster together exactly, following the hsrω-n transcripts. Immunoprecipitation studies using the hnRNP antibodies further demonstrated a physical association of hnRNPs and hsrω transcripts. The omega speckles are distinct from interchromatin granules since nuclear speckles containing serine/arginine-rich SR-proteins like SC35 and SRp55 did not colocalize with the omega speckles. The speckled distribution of hnRNPs was completely disrupted in hsrω nullosomics. We conclude that the hsrω-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.