The cell nucleus is organized as discrete domains, often associated with specific events involved in chromosome organization, replication, and gene expression. We have examined the spatial and functional relationship between the sites of heat shock gene transcription and the speckles enriched in splicing factors in primary human fibroblasts by combining immunofluorescence and fluorescence in situ hybridization (FISH). The hsp90α and hsp70 genes are inducibly regulated by exposure to stress from a low basal level to a high rate of transcription; additionally the hsp90α gene contains 10 introns whereas the hsp70 gene is intronless. At 37°C, only 30% of hsp90α transcription sites are associated with speckles whereas little association is detected with the hsp70 gene, whose constitutive expression is undetectable relative to the hsp90α gene. Upon exposure of cells to heat shock, the heavy metal cadmium, or the amino acid analogue azetidine, transcription at the hsp90α and hsp70 gene loci is strongly induced, and both hsp transcription sites become associated with speckles in >90% of the cells. These results reveal a clear disconnection between the presence of intervening sequences at specific gene loci and the association with splicing factor–rich regions and suggest that subnuclear structures containing splicing factors are associated with sites of transcription.