Thiamine pyrophosphate (TPP) is a required cofactor in Salmonella typhimurium that is generated de novo by the condensation of 4-amino-5-hydroxymethyl pyrimidine (HMP) pyrophosphate and 4-methyl-5-(beta-hydroxyethyl)-thiazole (THZ) monophosphate. The THZ and HMP moieties are independently synthesized, and labeling studies have demonstrated probable metabolic precursors to both. We present herein the initial characterization of thiI, a gene required for THZ synthesis. We show that thiI is a 1,449-bp open reading frame located at minute 9.65 on the S. typhimurium chromosome and that it encodes a 483-amino-acid protein with a predicted molecular mass of 55 kDa. Unlike genes in the thiamine biosynthetic operon at minute 90, thiI is not transcriptionally regulated by TPP.