Aqueous solutions of the triblock copolymer poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) EO13PO30EO13 (Pluronic L64) were investigated over a wide concentration range (20-100%, (w/w) polymer) in the micellar, liquid crystalline and reverse micellar phases, using electron spin resonance (ESR) spectroscopy of spin probes. A series of amphiphilic nitroxide spin probes based on n-doxyl-stearic acid (nDSA) with n, the carbon atom to which the doxyl group is attached, equal to 5 and 10 were used to measure the local polarity, dynamics and degree of order in the self-assembled system. The N-14 isotropic hyperfine splitting, alpha(N), was the polarity sensitive parameter. The probe location and the corresponding effective local hydration, Z(eff), were deduced by comparing ESR spectra of the probes in L64 solutions with spectra of the probes in aqueous solutions of poly(ethylene oxide) (PEO), poly(propylene oxide) (PPO) and a mixture of PEO and PPO containing the monomer molar ratio 26:30, as in L64. The results indicate that the probes reside in and provide evidence for the presence of hydrophobic and non-hydrated regions consisting of PO blocks and that the order in the aggregates decreases from the PO/EO interface toward the PO domains. Additional support for these conclusions was obtained from ESR spectra of the probes in the lamellar phase as a function of added cholesterol and by simulations of the ESR spectra of the probes in the lamellar phase of L64. This study of the hydrophobic part of the aggregates, together with our previous study based on ESR spectra of cationic probes that reside in the polar and hydrated EO regions, lead to a detailed description of the nature of L64 aggregates in aqueous solutions. (C) 2000 Elsevier Science Ltd. All rights reserved.