Purpose. To investigate the suitability of three antimicrobial peptides (AMPs) as cellpenetrating antimicrobial peptides. Methods. Cellular uptake of three AMPs (PK-12-KKP, SA-3 and TPk) and a cellpenetrating peptide (penetratin), all 5(6)-carboxytetramethylrhodamine-labeled, were tested in HeLa WT cells and analyzed by flow cytometry and confocal microscopy. Furthermore, the effect of the peptides on eukaryotic cell viability as well as their antimicrobial effect were tested. In addition, the secondary structure and disrupting ability of the peptides in the presence of bilayer membranes of different composition were analyzed. Results. AMP uptake relative to penetratin was ~13% (PK-12-KKP), ~66% (SA-3) and ~50% (TPk). All four peptides displayed a punctate uptake pattern in HeLa WT cells with co-localization to lysosomes and no indication that clathrin-mediated endocytosis was the predominant uptake mechanism. TPk showed the highest anti-bacterial activity. Only penetratin displayed clear secondary structure in the presence of membrane-mimicking liposomes. SA-3 exhibited selective disruption of liposomes mimicking Gram-positive and Gram-negative membranes. Conclusion. PK-12-KKP is an unlikely candidate for targeting intracellular bacteria, as the eukaryotic cell-penetrating ability is poor. SA-3, affected the cellular viability to an unacceptable degree. TPk showed acceptable uptake efficiency, high antimicrobial activity and relatively low toxicity, and it is the best potential lead peptide for further development.