We develop second-harmonic nanoparticles as the contrast agents for cell imaging. Second-harmonic nanoparticles show promise as cell imaging probes due to their non-bleaching, non-blinking, and coherent signal. Nanoparticles of noncentrosymmetric crystal structures have high second-harmonic generation (SHG) efficiency and provide high contrast in a generally non-structured cell environment. Here, we use barium titanate (BaTiO3) nanoparticles with tetragonal crystal structure as imaging probes. Cytotoxicity tests performed on BaTiO3 nanoparticles with mammalian cells did not result in toxic effects. Specifically we observed no change in the cell metabolism after 24 hours incubation of the cells with high concentration of BaTiO3 nanoparticles. We demonstrate two methods of cell labeling with BaTiO 3 nanoparticles for imaging. One is non-specific labeling via endocytosis of the cells, which results in a great number of the nanoparticles randomly distributed inside the cells. The other is specific labeling via surface functionalization of the nanoparticles with antibodies, which enables us to label specific cell membrane proteins with the nanoparticles. SHG imaging is compatible to two-photon microscopy and the SHG signal from nanoparticles can be easily detected with a standard two-photon confocal microscope. Our work provides the opportunity for long-term, three-dimensional cell tracking with secondharmonic nanoparticles. © 2010 SPIE.