Temperature-induced intracellular uptake mechanism of thermoresponsive polymeric micelles comprising poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)-b-poly(d,l-lactide) (P(IPAAm-DMAAm)-b-PLA) inside cultured bovine carotid endothelial cells is investigated by flow cytometry and confocal laser scanning microscopy. Hydrodynamic sizes of P(IPAAm-DMAAm)-b-PLA micelles are approximately 20 nm below the lower critical solution temperature (LCST) of 39.4 degrees C, and their sizes increased to ca. 600 nm above the LCST due to the aggregation of micelles. Intracellular uptake of P(IPAAm-DMAAm)-b-PLA micelles is significantly limited at a temperature below the micellar LCST, 37 degrees C. Of great interest, the P(IPAAm-DMAAm)-b-PLA micelles are internalized into the cells above the micellar LCST (42 degrees C), being dependent on polymer concentration, time, and temperature. By contrast, no intracellular uptake of polyethylene glycol-b-PLA micelles is observed regardless of temperature changes. Enhanced intracellular micelle uptake is probably due to the enhanced interactions between the micelles and cell membranes through the dehydration of corona-forming thermoresponsive polymer chains. Internalization of submicrometer-scale micellar aggregates inside the cells is probably due to their various endocytosis mechanisms. P(IPAAm-DMAAm)-b-PLA micelles localize at the Golgi apparatus and endoplasmic reticulum, but not inside lysosomes. These results indicate that the thermoresponsive polymeric micelles are greatly promising as intracellular delivery tools of drugs, nucleic acids, and peptides/protein without lysosomal decomposition in conjunction with applied heating.