The alternative pyrimidine biosynthetic (APB) pathway can synthesize the 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) moiety of thiamine in Salmonella typhimurium independently of de novo purine biosynthesis. When mutants defective in function of the APB pathway were isolated, the predominant class (40%) were defective in a single locus we have designated apbC. Mutations in apbC block function of the APB pathway since they prevent growth of a purF mutant in the absence of thiamine. Lesions in apbC also cause a thiamine auxotrophy in strains proficient in purine biosynthesis when fructose is provided as the sole carbon and energy source. Results presented here are consistent with ApbC being involved in the conversion of aminoimidazole ribonucleotide to HMP, and we suggest that ApbC performs a redundant step in thiamine synthesis. Sequence analysis demonstrated that apbC mutations were alleles of mrp, a locus previously reported in Escherichia coli as a metG-related protein. We propose that this locus in S. typhimurium be designated apbC to reflect its involvement in thiamine synthesis.