The tumour targeting properties of a new drug carrier synthesised by bioconjugation of folic acid to beta-cyclodextrins through a PEG spacer (CD-PEG-FA) were investigated. Surface plasmon resonance demonstrated that CD-PEG-FA interacts specifically with immobilized folate binding protein (FBP) with apparent dissociation constant of 3.59 nM while the naked beta-cyclodextrins did not display any specific interaction. Cell culture studies showed that CD-PEG-FA was devoid of cell toxicity. [3H] folic acid/CD-PEG-FA competition binding investigations performed with over-expressing folate receptor human epidermal carcinoma KB cells showed that CD-PEG-FA had about 10 times lower tumour cell binding capacity than folic acid free. The carrier cell trafficking properties were investigated using rhodamine-B as fluorescent probe, which possesses 3000 M-1 and 4600 M-1 inclusion constants for CD-PEG-FA and beta-cyclodextrins, respectively. Cell associated fluorescence measurements showed that CD-PEG-FA did not promote the rhodamine-B uptake into non-folate receptor expressing human lung carcinoma MCF7 cells while 19% higher accumulation in KB cells was found with respect to native beta-cyclodextrins. Confocal laser scanning microscopy evidenced the presence of cytosolic red fluorescent spots after 2 hours incubation of KB cells with rhodamine-B CD-PEG-FA inclusion. The fluorescent dye resided primarily into small spots, namely endosomes and multi-vescicular bodies. At 1 hour after pulsed incubation wider red fluorescent cellular structures appeared as fusion of previous structures.