The biosynthetic pathways of the thiazole moiety of thiamin were studied in the archaeon Halobacterium salinarum. Thiamin is generated by the union of 4-amino-5-hydroxymethyl-2-methylpyrimidine (pyrimidine) and 5-(2-hydroxyethyl)-4-methylthiazole (thiazole). The biosynthesis of thiazole is different in facultative anaerobes, aerobes and eukaryotes. In eukaryotes, the C-4, -4', -5, -5' and -5" of the thiazole is biosynthesized from nicotinamide adenine dinucleotide (NAD), with cysteine as S donor and the C-2 and N atoms of glycine. In facultative anaerobic bacteria, such as Escherichia coli, the precursors of the thiazole are the N and C-2 atoms from tyrosine and C-4, -4', -5, -5' and -5" from 1-deoxy-D-xylurose-5-phosphate, again with cysteine as S donor. In aerobic bacteria, such as Bacillus subtilis, L-tyrosine is replaced by glycine. In Archaea, known as the third domain of life, the biosynthetic pathway of thiamin has not yet been elucidated. In the present study in the archaeon H. salinarum, it was shown that both the N and C-2 from glycine are incorporated into the thiazole, rather than the N atom coming from L-tyrosine. These results show that thiazole biosynthesis in H. salinarum more closely resembles the biosynthetic pathway found in eukaryotes.