The first five steps in de novo purine biosynthesis are involved in the formation of the 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) moiety of thiamine. We show here that the first enzyme in de novo purine biosynthesis, PurF, is required for thiamine synthesis during aerobic growth on some but not other carbon sources. We show that PurF-independent thiamine synthesis depends on the recently described alternative pyrimidine biosynthetic (APB) pathway. Null mutations in zwf (encoding glucose-6-dehydrogenase), gnd (encoding gluconate-6-P dehydrogenase), purE (encoding aminoimidazole ribonucleotide carboxylase), and purR (encoding a regulator of gene expression) were found to affect the function of the APB pathway. A model is presented to account for the involvement of these gene products in thiamine biosynthesis via the APB pathway. Results presented herein demonstrate that function of the APB pathway can be prevented either by blocking intermediate formation or by diverting intermediate(s) from the pathway. Strong genetic evidence supports the conclusion that aminoimidazole ribotide (AIR) is an intermediate in the APB pathway.