In this study, a mouse gene and cDNA encoding for a novel skeletal muscle-specific choline transporter-like protein 1 (mCTL1) were identified and mCTL1 mRNA and protein expression characterized. The mCTL1 cDNA is 2888-bp long; consisting of a 653-amino-acid open-reading frame, 8-11 putative transmembrane domains, three N-glycosylation sites and seven protein kinase C phosphorylation sites. The mCTL1 gene is localized to chromosome 4B2, at 182 kb in length, and encoded by 17 exons. Although the mCTL1 mRNA was expressed in several mouse tissues such as muscle, brain, heart and testis, the protein analyses of multiple tissues and membrane vesicles reveal that mCTL1 is exclusively expressed in skeletal muscle. Expression of His-tagged mCTL1 in Cos-7 cells produces an increase in saturable choline uptake that is sensitive to a Na(+)-ion gradient, ethanolamine and the Ca(2+)-channel blocker verapamil, and insensitive to low concentrations of hemicholinium-3.