The isotopes of [6-13C]- and [5'-2H2]pyridoxine were incorporated efficiently into the pyrimidine moiety of thiamin in S. cerevisiae. The mass fragmentation pattern showed that the C-6 and H-5' atoms of pyridoxine were incorporated into the C-6, and H-5' atoms of the pyrimidine, respectively. These findings, along with out previous results, show that the C-2', C-2, N-1, C-6, C-5, and C-5' unit of the pyrimidine are derived from the C-2', C-2, N-1, C-6, C-5 and C-5' unit of pyridoxine, and that pyrimidine is biosynthesized from pyridoxine and histidine.