proteins implicated in choline transport for phospholipid synthesis. In this study we characterized the 5'-flanking region of the human hCTL1 gene and examined some of the possible mechanisms of its regulation including promoter activity, splicing and expression. The transcription start site of the hCTL1 gene was mapped by 5'-RACE and the presence of two splice variants, hCTL1a and hCTL1b, was investigated using isoformspecific PCR and 3'-RACE. The hCTL1 promoter region of ~900 bp was isolated from MCF-7 human breast cancer cells. The promoter is TATA-less and driven by a long stretch of GC-rich sequence in accordance with a widespread expression of hCTL1 at both mRNA and protein levels. Deletion analyses demonstrate that a very strong promoter is contained within 500bp of the transcription start site and more upstream regions are not increasing its activity. The core promoter that confers the minimal transcription is within the -188bp/+27bp region and its activity varies in human breast cancer and mouse skeletal muscle cells. Multiple motifs within the promoter regulatory region bind nuclear factors from both cultured cells and normal human skeletal muscle. The motifs within the three regions, S1 (-92/-61bp), S2 (-174 / -145bp), and S3 (-289 /-260bp), contain overlapping binding sites for the haematopoietic transcription factors and the ubiquitous transcription factors in line with expected gene function. Genomic analyses demonstrate a high conservation of the human and mouse CTL1 proximal promoters. Accordingly, mRNA profiles demonstrate that human splice variants are expressed ubiquitously as demonstrated for the mouse transcripts, however they differ from the profiles of the rat CTL1 transcripts, which is more restricted to the neurons and intestinal tissues. The shorter hCTL1b variant contains the cytosolic C-terminal motif L⁶⁵¹KKR⁶⁵⁴ for endoplasmic reticulum retrieval/retention. This retention signal is conserved in the human, rat and mouse CTL1b and typical for the trans-membrane proteins of type 1 topology.