Nuclear magnetic resonance (NMR) spectroscopy has been employed for structural characterization of daunorubicin-loaded poly(butylcyanoacrylate) nanoparticles. Measurements of the nuclear relaxation times (T-1) and application of diffusion ordered spectroscopy (DOSY), obtained through pulsed field gradient (PFG) NMR experiments, have been performed to determine the supramolecular structure of the drug-polymer conjugates and to clarify the mechanisms of drug immobilization in the polymer matrix. The results confirm the coexistence of three different interaction mechanisms between the drug daunorubicin and the poly(butylcyanoacrylate) nanopartides, viz., (i) drug association with the polymer chains, through H-bonds and/or dipole charge interactions, (ii) physical entrapment of the drug within the polymer matrix, and (iii) drug adsorption on the surface of nanoparticles. The identification of these three different interaction mechanisms suggests that drug; delivery using the nanoparticle vehicle has potential for sustained.delivery/release of daunorubicin.