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This paper examines the exocytosis of nanoparticles, an important but rarely documented aspect of cell responses to engineered nanomaterials. Specifically, HT-29 mammalian cells were labeled with a lipophilic Laurdan dye, and the membrane fluidity in the presence of gold nanoparticles was evaluated by the generalized polarization (GP) values derived from the fluorescence spectra of the dye. The concentration of excreted gold nanoparticles increased with increasing extracellular calcium, directly demonstrating exocytosis of nanoparticles and corroborating the inference made from the GP values of the Laurdan dye. This study provides additional information for understanding the fate of nanomaterials in biological systems.