The color observed in the Jaffe reaction is due to the formation of the isomers of the 1 : 1 σ-complex (I) of creatinine and picric acid. The formation of hydroxylated anions of I (I') and the presence of equilibria between the isomers of I and I' were confirmed by nuclear magnetic resonance studies. The phenomenon of the augmentation of the color intensity on neutralizing the alkaline reaction mixture of picric acid and creatinine can be explained in terms of shifts of the equilibria between the isomers of I and I'. Three isomers of the 1 : 2 σ-complex are formed via the isomers of I and I' in an alkaline mixture of picric acid and creatinine, but not in the presence of a large excess of picric acid, as determined by high performance liquid chromatography.