Cells internalized synthetic oligonucleotides (oligos) in culture. The hybridization of these molecules to target RNA in the living cell was subsequently detected and characterized after fixation of the cells, with or without previous detergent extraction. Hybridized oligo was distinguished from free oligo in the cell using an in situ reverse transcription technique. This assay exploited the ability of the hybridized oligo to prime synthesis of a specific cDNA strand; unhybridized oligo present in the cell could not act as a primer for reverse transcription. Phosphorothioate and fluorochrome-labeled phosphodiester oligo dT were found to enter cells rapidly and hybridize to poly (A) RNA within 30 min. Hybrids containing phosphorothioate oligo dT were detectable in cells after up to 4 h of efflux time. Phosphodiester bonded oligo dT containing covalently-linked fluorochromes appeared more stable in the cell than unmodified phosphodiester oligo dT; hybrids containing these oligos could be detected in cells as long as 18h after efflux began. The in situ transcription assay was also sensitive enough to detect hybridization of anti-actin oligos to actin mRNA in the cell. It is probable, therefore, that this assay can be used to help assess the efficacy of antisense oligos by their hybridization to a target mRNA in cells or tissues; hybridized oligos are more likely to induce a specific antisense effect. Additionally, this assay will help to identify probes that would be useful as stable hybridization tags to follow RNA movement in living cells.