Pyrimidinyl-cysteine, which was produced from thiamine and cysteine by the catalytic exchange reaction of a purified bacterial thiaminase I. was isolated as a crystal, mp. 219-220℃ (decomp.), by the column chromatography using Amberlite IRC-50. The yield was 61%. The crystal showed positive reaction with Dragendorff or ninhydrine reagents, but negative with nitroprusside reaction. Nitroprusside reaction did not appear even after the treatment of the crystal by zinc in acid solution. Therefore, the chemical construction of pyrimidinyl-cysteine was postulated as S-(2-methyl-4-aminopyrimidinyl-5-methyl)-cysteine. The results of elemental analysis of C, H, N and S in the compounds obtained enzymatically and chemically were almost identical with the theoretical value of the formula.