The endocytotic mechanisms involved in the uptake of charged polystyrene nanoparticles into HeLa cells were investigated. Uptake experiments were done in the presence or absence of drugs known to inhibit various factors in endocytosis. Independent of the particle charge, endocytosis is highly dependent on dynamin, F-actin, and tyrosine-specific protein kinases, which suggests a dynamin-dependent and lipid raft-dependent mechanism. However, cholesterol depletion did not hinder particle uptake. Regarding positively charged particles, macropinocytosis, the microtubule network, and cyclooxygenases are also involved. The clathrin-dependent pathway plays a minor role.