In prokaryotes, C-atom of formate is incorporated into the C-2 atom of the pyrimidine moiety of thiamin, while in eukaryotes, it is incorporated into the C-4 atom. We confirmed the incorporation of the C-atom of formate into the C-4 atom of the pyrimidine portion in mitochondria as well as in cytosol of Saccharomyces cerevisiae, which means, the thiamin in mitochondria was biosynthesized via the eukaryotic biosynthetic pathway, not the prokaryotic pathway. While the question remains of whether the thiamin in mitochondria was biosynthesized in situ or transported from the cytoplasm, we were able to confirm that the thiamin in mitochondria of S. cerevisiae was synthesized via eukaryotic pathway.