The major limitation in the application of bioactive molecules is their low permeation across plasma membrane. Effective transporters - cell-penetrating peptides (CPPs) - are utilized to enhance uptake of various cargo upon attachment to its sequences. Still, information about relevance of different endocytic routes during CPP-cargo internalization is ambiguous and underlying mechanism(s) of intracellular trafficking is even less understood. We first defined involvement of recycling pathway in trafficking of 3 different CPPs - transportan, oligoarginine and Tat - complexed to avidin-TexasRed in Cos-7 cells in relation to trans-Golgi network spatially constraining recycling endosomes. By confocal microscopy, only a negligible fraction of complexes-containing vesicles were found inside trans-Golgi ring suggesting its marginal role in CPP-mediated delivery. Secondly, we characterized engagement of endo-lysosomal pathway to assess acidity of complexes-containing vesicles. CPPs induced 3 different populations of complexes-containing vesicles which size and proportion depended on CPP, time and concentration. In time, more complexes were targeted to low-pH structures. However, a population of complexes-containing vesicles was observed to retain rather neutral pH. Induction of vesicles with non-acidic pH generated i.e. by caveolin-dependent endocytosis or by CPPs themselves during intracellular trafficking could be the key step in inducement of escape of complexes from endosomal structures, a limiting step in effective cargo delivery by CPPs.