In actively transcribing cells, factors involved in pre-mRNA splicing localize in a speckled pattern at the fluorescence microscopic level. The speckled pattern corresponds to interchromatin granule clusters and perichromatin fibrils at the electron microscopic level. Based upon [3H]uridine incorporation studies transcription is thought to occur at the perichromatin fibrils and not within interchromatin granule clusters. We have shown that upon inhibition of RNA polymerase II transcription or pre-mRNA splicing, splicing factors redistribute and preferentially localize to interchromatin granule clusters, which become larger and more uniform in shape. Introduction of exogenous DNA templates into the cell nucleus results in a recruitment of splicing factors to the new sites of transcription. These data suggest that splicing factors are localized at storage and/or reassembly sites (interchromatin granule clusters) and are recruited to active sites of transcription (perichromatin fibrils). Furthermore, these data demonstrate that the speckled localization of pre-mRNA splicing factors is a reflection of the transcriptional and pre-mRNA splicing activities of the cell.