We have previously reported the cDNA cloning of the mouse thiamine transporter THTR-1 as a p53 transcriptional target gene (renamed THTR-1a hereinafter). The mouse THTR-1a is predicted to encode a protein of 12 hydrophobic stretches and a hydrophilic loop of 87 amino acids between transmembrane helices VI and VII. The mouse THTR-1 gene has been cloned, two major transcriptional start sites located at -175 and -183 relative to the translation start codon were identified. In addition, we have cloned a spliced variant, designated THTR-1b, from mouse liver cDNA library. This isoform is characterized by an inframe deletion of 114 nucleotides from the 3'-terminal region of exon 2, predicting the expression of a truncated protein lacking the central 38 amino acids of the loop region. THTR-1b coexpressed with THTR-1a in many of the mouse tissues and in day-7 to day-17 embryos, but in lower levels than the THTR-1a. When expressed in mammalian cells, both isoforms were able to mediate the transport of thiamine. Therefore, the transport function of the mouse THTR-1 is not determined by the central 38 amino acids of its loop region.