Self-association of metal-free human insulin in aqueous solution at pH 9.4 has been studied using pulsedfield gradient (PFG) NMR spectroscopy. The diffusion coefficients were measured for two different insulin concentrations, (0.72 \mathrm{~mm}) in which the dimer is the predominant species and (3 \mathrm{~mm}) which is a mixture of dimer and tetramer. In the more concentrated solution, exchange between the two aggregates is slow on the NMR chemical shift time scale. As a result, the dimer and tetramer give rise to separated resonances in the aromatic region of the ({ }^{1} \mathrm{H}) NMR spectrum. The diffusion coefficient determined for the tetramer by PFG NMR is (1.07 \times 10^{-6} \mathrm{~cm}^{2} \mathrm{~s}^{-1}) at (298 \mathrm{~K}), while that for the dimer is (1.38 \times 10^{6} \mathrm{~cm}^{2} \mathrm{~s}^{1}). The hydrodynamic diameters calculated for the tetramer and dimer are 46 and (36 \AA), respectively, which are in good agreement with those measured by dynamic light scattering. The advantage of PFG NMR for the measurement of insulin aggregation in these solutions results from the chemical shift selectivity which allows diffusion coefficients to be directly calculated for the different aggregates. The (T_{1}) and (T_{2}) relaxation times of the dimer and tetramer aromatic protons were also measured in order to optimize the parameters of the PFG NMR experiment and correctly interpret the results of the measured spectra. 1995 Academic Press, Inc.