The yeast Saccharomyces cerevisiae possesses a choline transport system encoded by the CTR gene. Incorporated choline is exclusively utilized for the synthesis of phosphatidylcholine. Determination of the complete nucleotide sequence of the CTR gene showed that the deduced primary sequence of the choline transporter comprised 563 amino acid residues with a calculated molecular weight of 62,055. Both the amino- and carboxyl-terminal regions were hydrophilic whereas the rest of the sequence was highly hydrophobic and contained several membrane-spanning regions. There were four potential glycosylation sites in the hydrophilic parts of the sequence. The transporter showed significant sequence similarities to the yeast arginine and histidine transporters. A 2.0-kilobase RNA species was identified as the CTR transcript. Disruption of the CTR locus completely abolished the choline transport activity, indicating that the CTR product is the sole choline transporter in yeast. The regulation of choline transport mRNA was investigated by Northern blot analysis using a CTR probe. The abundance of CTR mRNA significantly decreased on incubation of cells with a combination of choline and myo-inositol. The CTR mRNA level was high in the exponential growth phase but decreased dramatically when cells entered the stationary phase. Similar control had been observed for some enzymes in yeast phospholipid synthesis.