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The ability of dendrimers to cross cell membranes is of much interest for their application in drug and gene delivery. Recent studies demonstrate that dendrimers are capable to enter cells by endocytosis, but the intracellular pathway following their internalization remains controversial. In this study we use confocal fluorescence microscopy to elucidate the intracellular trafficking properties of PAMAM dendrimers with high spatial and temporal resolution in living HeLa cells. Macromolecules of different chemical functionality (neutral, cationic and lipidated), size (from G2 up to G6) and surface charge are investigated and their internalization properties correlated with the molecular structure. Toxicity and internalization data are discussed that allow the identification of dendrimers maximizing intracellular uptake with the minimum effect on cell viability. Time-lapse imaging and colocalization assays with fluorescent biomarkers for endocytic vesicles demonstrate that dendrimers are internalized by both clathrin-dependent endocytosis and macropinocytosis and are eventually delivered to the lysosomal compartment. Moreover we analyzed the uptake of dendrimers in additional cell lines of practical interest for therapeutic purposes. These measurements together with a direct comparison with TAT peptides demonstrate that PAMAM dendrimers possess similar properties to these widely used cell-penetrating peptides and thanks to their chemical tunability may represent a valid alternative for drug and gene delivery.