Twelve guanidino compounds including, among others, glycocyamine, creatine, creatinine, arginine, guanidinosuccinic acid were submitted to thin-layer chromatography on plates precoated with silica gel or cellulose, respectively. Optimal resolution was obtained by two-dimensional chromatography on cellulose layers when the two solvent systems iso-propanol-acetic acid-water (volumes, 60 ml + 45 ml + 15 ml) and iso-propanol-dimethyl-formamide-water (volumes, 40 ml + 20 ml + 20 ml) were used for the first and second dimension, respectively. This procedure permitted the resolution of 10 distinct spots out of a mixture of the twelve guanidino compounds. Chloroform-methanol-ammonia (170 g/kg) (volumes, 40 ml + 20 ml + 20 ml) proved to be the most suitable solvent system for one-dimensional chromatography on cellulose layers of less complex mixtures of guanidino compounds.