Transketolase from baker’s yeast is a thiamin diphosphate-dependent enzyme in sugar metabolism that reconstitutes with various analogues of the coenzyme. The methylated analogues (4¢-methylamino-thiamin diphos-phate and N1¢-methylated thiamin diphosphate) of the native cofactor were used to investigate the function of the aminopyrimidine moiety of the coen-zyme in transketolase catalysis.