Mesoporous silica nanoparticles (MSN) were functionalised by aminofluorescein (AMF) with diethylenetriaminepentaacetic acid spacer molecules which provide free carboxylic groups for binding cell-specific ligands such as folate. AMF allowed the exploration of cellular uptake by HeLa cells using confocal microscopy and flow cytometry. The functionalized nanoparticles (MSN-AMF) penetrated efficiently into HeLa cell cytoplasm through a clathrin dependent endocytosis mechanism. The number of endocytosed MSN-AMF was enhanced when using folate as a targeting molecule. Uptake kinetics revealed that most of MSN-AMF were internalized within 4 h of incubation. Moreover, we found that MSN-AMF were capable of escaping the acidic endolysosomal vesicles of HeLa cells. Cytotoxicity studies suggested that these nanoparticles are non-toxic to HeLa cells up to a dose level of 50 microg/ml.