Radioactivity from [¹⁴C]formate, from D-[1-¹⁴C]-, D-[2-¹⁴C]-, and D-[6-³H,6-¹⁴C]glucose, from D-[1-¹⁴C]fructose and from [1,3-¹⁴C]- and [2-¹⁴C]glycerol is incorporated nonrandomly into the pyrimidine moiety of thiamin in Saccharomyces cerevisiae. The observed incorporation pattern, established by a new chemical degradation, leads to the inference that there are two biosynthetic pathways to the pyrimidine moiety of thiamin in yeast. In the major pathway, formate is the precursor of C-4 of the pyrimidine nucleus, while hexose metabolites serve as the source of the remaining five carbon atoms of the pyrimidine unit. In the minor pathway, it is C-2 and not C-4 of the pyrimidine nucleus which is derived from formate, while C-4,-5 originates from carbohydrate. The source of C-2', C-Y, and C-6 of the pyrimidine unit in this minor pathway remains unknown. Activity from [ureido-¹⁴C]citrulline and [¹⁴C]urea is not incorporated into thiamin.