The interaction of the osmolytes trimethylamine N-oxide (TMAO) and urea in aqueous solutions at 40 °C was investigated by isotopic substitution neutron scattering at a TMAO mole fraction of 0.05 and TMAO/urea concentration ratios of 1 : 2 and 1 : 4. The partial pair distribution functions obtained by the empirical potential structure refinement method are consistent with those obtained previously for similar pure TMAO and 1 : 1 TMAO-urea solutions and indicate that urea progressively replaces the water molecules in the first coordination shell of the TMAO oxygen atom. The apparent association constant for the TMAO : urea complex (K(1)) was calculated to be 0.14 M(-1), which is of the same order as the experimental urea-protein binding constants per site reported in the literature. This confirms that the two osmolytes act independently at least in the physiological range.