By using fluorescent in situ hybridization (FISH), we visualized viral RNA of human rhinovirus type 2 (HRV2) during its entry into HeLa cells. RNA uncoating of HRV2 is entirely dependent on low endosomal pH (< or =5.6). When internalized into cells treated with bafilomycin, which results in neutralization of the endosomal pH, no FISH signal was recorded, whereas in the absence of the drug, fluorescent dots were seen. Therefore, FISH detects the genomic viral RNA only upon its release from the capsid. Free viral RNA was first seen at 10 min postinfection (p.i.) in the perinuclear area of the cell, which is indicative of RNA release in/from late endosomal compartments. Pulse-chase experiments and observation of HRV2 RNA and capsid proteins via microscopy, Western blotting, and reverse transcription-PCR revealed that the RNA signal persisted whereas the protein signal disappeared. This demonstrates transport of capsids to lysosomes and degradation. In contrast, viral RNA that had already been transferred into the cytoplasm escaped lysosomal breakdown as indicated by a persistent FISH signal. Taken together, our results demonstrate by direct means RNA arrival in the cytosol within 10 min p.i. Based on persistence of the FISH signal and productive infection in the presence of the microtubule-depolymerizing drug nocodazole, we localized this process to endosomal carrier vesicles/late endosomes.