Thiamin phosphate synthase couples 4-amino-5-(hydroxymethyl)-2-methylpyrimidine pyrophosphate (HMP-PP) and 4-methyl-5-(beta-hydroxyethyl)thiazole phosphate (Thz-P) to make thiamin phosphate on the biosynthetic route to thiamin-PP, the active form of vitamin B-1. We show that this coupling reaction goes through a pyrimidine carbocation intermediate by using transient state kinetic methods, and that the rate constant for carbocation formation at the active site of the enzyme is 0.4 s(-1).