Quantum dots (QDs) are fluorescent semiconductor nanocrystals with superior optical properties compared to organic dyes currently undergoing rapid development for biological applications, particularly in fluorescence imaging. The folate receptor, overexpressed in a broad spectrum of malignant tumors, is an attractive target for selective delivery of imaging agents to tumor cells. This study examines nanoparticles containing QDs entrapped in a lipid shell, and post-loaded with a folate-lipid conjugate for targeting to mouse and human tumor cells expressing the folate receptor. Hydrophobic QDs were mixed with 1,2 dipalmitoyl-sn-glycero-3 phosphocholine and methoxy-polyethylene-glycol-distearoyl-phosphatidyl-ethanolamine (mPEG-DSPE) generating a nanoparticle referred to as lipodot, with a mean diameter size of approximately 100 nm. Folate-derivatized PEG-DSPE was post-loaded into the lipodots at 0.5% lipid molar concentration. Mouse J6456 lymphoma cells (J6456-FR) and human head and neck KB cancer cells (KB-FR), up-regulated for their folate receptors, were incubated with folate-targeted and non-targeted lipodots in vitro. Using fluorescence microscopy, it was found that only folate-targeted lipodots were taken up by tumor cells. Confocal depth scanning showed substantial internalization. Confirming the specificity of folate-targeted lipodots, binding and internalization were inhibited by free folate, and no uptake was found in a folate-receptor negative cell line. Selective binding and uptake of folate-targeted lipodots by J6456-FR cells was also observed in vivo after intra-peritoneal injection in mice bearing ascitic J6456-FR tumors based on FACS analysis and confocal imaging of harvested cells from the peritoneal cavity. Folate-targeted lipodots represent an attractive approach for tumor cell labeling both in vitro and in vivo.