In the mammalian cell nucleus pre-mRNA splicing factors are organized in a speckled pattern. The fluorescence signal within speckles appears homogeneous when cells are immunolabeled with antibodies directed against pre-mRNA splicing factors and examined by fluorescence microscopy. We have reexamined the speckled domains using serial dilutions of antibodies against SR proteins, snRNPs, and a 3' end processing protein by immunofluorescence and confocal laser scanning microscopy. Using higher antibody dilutions, the speckled domains consist of numerous subdomains that are spherical. and heterogeneous in size ranging from 0.2 to 0.5 mu m in diameter. We refer to these subdomains as "subspeckles." Each speckle is composed of 5 to 50 subspeckles and in some cases in actively transcribing cells, strings and loops of subspeckles were observed to extend from the speckled domains. Upon inhibition of RNA polymerase II transcription, the strings and loops of subspeckles were no longer observed. Subspeckles were also not observed in coiled bodies. Using fluorescence in situ hybridization we found subspeckles to be colocalized with transiently expressed beta-tropomyosin RNA transcripts. The compartmentalization into subspeckles may represent an efficient way of organizing these factors for their subsequent transport to transcription/RNA processing sites.