The nuclear localization of various cell penetrating peptides (CPPs), including Tat [47-57], YG(R)9, YG(K)9, and model amphipathic peptide (MAP), was examined and correlated with the endocytosis and cytosolic transfer efficiency in CHO cells. The results showed that the internalization of the amphipathic peptide, MAP, was much higher than that of the other cationic CPPs tested. During subcellular fractionation analysis, MAP was only found in the vesicular fraction and was not detectable in the cytosol, similar to the intracellular localization of YG(K)9 as previously determined. This localization pattern differs greatly from the cationic CPPs oligoarginine and Tat, which were previously found primarily in the cytosol. Both quantitative and qualitative analysis of MAP showed high nuclear localization, with staining in perinuclear vesicles. On the other hand, YG(R)9 was found to be excluded from the nucleus. Lysosomotropic amines altered the nuclear localization of the CPPs tested, and the change was correlated with the release of degradation products from the treated cells. These results suggest that highly endocytosed CPPs such as MAP may be more suitable for nuclear drug delivery applications than peptides such as Tat and YG(R)9 that are efficiently delivered to the cytosol.