The folate receptor (FR) is a highly selective tumor marker over expressed in many human cancers and it constitutes a useful target for tumor-specific drug delivery. Thus, the conjugation of folic acid to different drugs or drug carriers may enhance the delivery of the therapeutic agent to FR-positive tumor cells. The aim of this study was to investigate the interactions of folate-conjugated polyalkylcyanoacrylate nanoparticles with tumor cells overexpressing the FR. For this purpose, nanoparticles were prepared by nanoprecipitation of the poly[aminopoly(ethylene glycol) cyanoacrylate-co-hexadecyl cyanoacrylate] [poly(H2NPEGCA-co-HDCA)] copolymer and labeled with the hydrophobic fluorescent dye nile red. Nile red-loaded nanoparticles were then conjugated to folic acid via the PEG terminal amino groups. Four human cancer cell lines were then tested by western blot in order to evaluate the FR expression levels. KB3-1 cell line showed the higher expression level, while MCF-7 cells were taken as a control. After measuring the cytotoxicity of the nanoparticles on these two cell lines, fluorescent folate-nanoparticles were incubated with them and the cellular uptake was evaluated by confocal microscopy and flow cytometry. KB3-1 cells showed a greater nanoparticle internalization, when compared to MCF-7 cells.