Nanoparticles have been studied extensively for over two decades however, much of the work has been focused on particle formulation and surface modification. While a significant amount of literature does exist on therapeutic application of these particles, somewhat limited information is available with regard to particle uptake, and intracellular trafficking of these unique delivery systems. In this work, the mechanisms of poly(lactide-co-glycolide) (PLGA) nanoparticle uptake and action were compared in epithelial cells of the airway, gut, and renal tubule in an attempt to fill this gap and decipher commonalities among the three different cell types. The amount of nanoparticle uptake by the cell, position of particles relative to intracellular compartments, and effect of particles on the bioelectric parameters of epithelial cells were determined. This data is particularly relevant to targeted nanoparticle design and therapeutic applications.