In Salmonella typhimurium, thiamine is a required nutrient that is synthesized de novo. Labeling studies have demonstrated probable precursors for both the 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate moiety and the 4-methyl-5-(beta-hydroxyethyl) thiazole monophosphate moiety. The isolation of thiamine auxotrophs with mutations in at least five different genetic loci is reported. The majority (22 of 25) of the mutants required only the thiazole moiety of thiamine to satisfy their growth requirement. Most (14 of 25) of the mutants were affected in the thi cluster at min 90 on the S. typhimurium genetic map. Data provided herein indicate that this cluster encodes an operon whose transcription is regulated by thiamine and suggest that thiamine pyrophosphate, or a molecule derived form it, is the effector molecule. Mutants with altered regulation of this operon were isolated, and we propose that they are defective in thiamine phosphate kinase, the product of the thiL gene.