The cytotoxicity and time-dependent membrane disruption by polypropylenimine dendrimer conjugates on cultured human umbilical vein endothelial cells (HUVEC) is reported. Fluorescently labeled derivatives of generation 5 polypropylenimine dendrimers were prepared via conversion of amines to acetamides or through the covalent attachment of high molecular weight poly(ethylene glycol) (PEG) chains. Direct interactions between the fluorescent dendrimer conjugates and HUVEC were monitored using confocal fluorescence microscopy to track dendrimer movement across the plasma membrane and the fluorescent staining of cell nuclei. Propidium iodide and lactate dehydrogenase cytotoxicity assays confirmed that chemical modification of the surface amines of the parental dendrimer to neutral acetamide or PEG functionalities eliminated their acute cytotoxicity. Cationic primaryamine-containing dendrimers demonstrated drastic time-dependent changes in the plasma membrane permeability and prominent cytotoxicity. However, complete removal of the primary amines or masking of the cationic surface via PEGylation decreased dendrimer cytotoxicity. Thus, preventing electrostatic interactions of dendrimers with cellular membranes apparently is a necessary step toward minimizing the toxicity of delivery vehicles to the endothelium.