The chemical composition of the water-soluble extracts of mature Cheddar cheese were identified, with the emphasis on understanding the interplay of compounds contributing to the savoury taste in Cheddar. The ultra-filtered water-soluble extracts of two mature Cheddar cheeses were fractionated by gel permeation chromatography (GPC). By sensory evaluation, two taste-active GPC fractions were identified from each cheese. On the basis of chemical profiling of these fractions, aqueous model tastant mixtures were prepared and sensory omission tests carried out. Glutamic acid, organic acids and mineral salts were the main tastants, whereas the other amino acids had a limited impact on taste. The characteristic umami taste was explained by a synergistic effect of glutamic acid and salts. Matching umami taste intensities were obtained from different concentrations of glutamic acid and salts. Unmasking of a bitter or sweet taste from mixtures of sub-threshold concentrations of amino acids without glutamic acids was also observed.