The precursor of the pyrimidine moiety of thiamin in S. cerevisiae was studied. The tracers [15N]- and [2'-13C]-pyridoxine were chemically synthesized, and incorporated efficiently into the pyrimidine. The mass fragmentation pattern showed that the nitrogen atom of pyridoxine was incorporated into the N-1 of the pyrimidine. In addition, the C-2' atom of pyridoxine was incorporated into the C-2' atom of the pyrimidine. These results and the structural resemblance between the pyrimidine and pyridoxine revealed that the C-2, C-2' and N-1 unit of the pyrimidine are derived from the C-2, C-2' and N-1 unit of pyridoxine.