Processing and intracellular transport of RNA transcripts are essential for gene expression. Translational regulation of gene expression may occur by several mechanisms, including control of transcript movement from sites of synthesis to site(s) of translation. We describe temporal analysis of the intracellular translocation of c-myc transcripts from the site of synthesis in the nucleus to sites of translation in the cytoplasm. Fluorescence in situ hybridization (FISH) and quantitative fluorescence microscopy were used to measure intracellular traffic of c-myc transcripts in individual recombinant cells following activation of c-myc sequences linked to a heat shock promoter. C-myc nuclear transcripts are visible in the nucleus within minutes of heat exposure. Transcripts remain in the nucleus for at least 4 hr after gene activation. Transport of c-myc transcripts to the cytoplasm begins -1 hr after cells are returned to 37Β°C. These data demonstrate the feasibility of measuring intracellular transcript transport following gene activation and provide a description of the kinetics of intracellular traffic of inducible c-myc transcripts in heated cells in situ.