Cell penetrating peptides (CPP) have been widely used to increase the cellular delivery of their associated cargo. Multiple modes of uptake have been identified; however, they cannot be predicted a priori. Elucidating these mechanisms is important for understanding peptide function as well as further optimizing cellular delivery. We have developed a class of mitogen activated protein kinase activated protein kinase 2 (MK2) inhibitor peptides, named FAK and YARA that utilize CPP domains to gain cellular access. In this study, we investigate the mechanism of endocytosis of these MK2 inhibitors by examining the uptake of fluorescently labeled peptide in human monocyte (THP-1) and mesothelial cells, and looking for colocalization with known markers of endocytosis. Our results indicate that uptake of the MK2 inhibitors was minimally enhanced by the addition of the fluorescent label, and that the type of endocytosis used by the inhibitor depends on several factors including concentration, cell type, and which CPP was used. We found that in THP-1 cells, the uptake of YARA occurred primarily via macropinocytosis, whereas FAK entered via all three mechanisms of endocytosis examined in this study. In mesothelial cells, uptake of YARA occurred via caveolae-mediated endocytosis, but became less specific at higher concentrations; whereas uptake of FAK occurred through clathrin-mediated endocytosis. In all cases, the delivery resulted in active inhibition of MK2. In summary, the results support endocytic uptake of fluorescently labeled FAK and YARA in two different cell lines, with the mechanism of uptake dependent on extracellular concentration, cell type, and choice of CPP. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.