The ninhydrin chromogenic reaction was used to determine the concentration of free amino groups in ripening Cheddar cheese. This assay involves dispersing cheese in citrate solution, heating an aliquot of the resulting mixture with aqueous dimethyl sulfoxide, ninhydrin plus lithium acetate solution at pH 5.2, diluting, and determining the absorbance at 570 nm. The ninhydrin assay and conventional amino acid analysis for lysine in freshly made Cheddar were in good agreement. There was a steady increase in the concentration of free amino groups as the cheese aged. The ninhydrin method was applied to several diverse cheese varieties and compared with the trinitrobenzenesulfonic acid procedure. Possible advantages of the ninhydrin assay are described.