Cell-penetrating peptides (CPPs) have been extensively used to deliver peptide nucleic acid (PNA) in cells. We have previously found that replacement of cytosine in triplex-forming PNAs with 2-aminopyridine (M) not only enhanced RNA binding, but also improved cellular uptake of PNAs. In this study, we used confocal fluorescence microscopy to evaluate the ability of CPPs to further improve cellular uptake of M-modified PNAs. We found that PNAs conjugated with Tat and octa-arginine peptides were effectively taken up in MCF7 cells when supplied in cell media at 1 μM. Remarkably, M-modified PNA without any CPP conjugation also showed strong uptake when the concentration was increased to 5 μM. Majority of PNA conjugates remained localized in distinct cytoplasmic vesicles, as judged by dot-like fluorescence patterns. However, M-modified PNAs conjugated with Tat, octa-arginine, and even a simple tri-lysine peptide also showed dispersed fluorescence in cytoplasm and were taken up in nuclei where they localized in larger vesicles, most likely nucleoli. Endosomolytic peptides or chemicals (chloroquine and CaCl2) did not release the conjugates from cytosolic vesicles, which suggested that the PNAs were not entrapped in endosomes. We hypothesize that M-modified PNAs escape endosomes and accumulate in cellular compartments rich in RNA, such as nucleoli, stress granules, and P-bodies.