The design, preparation and characterization of poly(butylcyanoacrylate) nanoparticles as a drug-delivery system for daunorubicin is reported. A range of light scattering [photon correlation spectroscopy (PCS)], spectroscopic [(1)H nuclear magnetic resonance ((1)H NMR), Fourier transform infrared (FTIR), chromatographic [gel permeation chromatography (GPC)] and quantum chemical techniques have been employed for the physicochemical characterization of drug-loaded nanoparticles and to clarify the mechanisms of drug immobilization in the polymer matrix. The presence of daunorubicin in the polymerization medium was found to affect both the degree of polymerization and the compactness of the resulting nanoparticles. The GPC, FTIR and (1)H NMR results confirmed cytostatic immobilization in the polymer matrix, with evidence for the presence of three types of inclusion: physically entrapped, polymer-associated (due to hydrogen bonds and/or dipole-charge interactions with the polymer chains), and polymer surface-adsorbed daunorubicin. The developed colloidal delivery system has the capacity for sustained in vitro release of daunorubicin. Preliminary in vitro assays were carried out on two cell lines, DLKP and DLKP-A, which display different levels of drug resistance, to evaluate the cytotoxicity of the drug-loaded nanoparticles.