The Na(+)- and Cl(-)-coupled betaine transporter, designated BGT1, a member of the neurotransmitter transporter gene family, is responsible for accumulation of betaine in hypertonic Madin-Darby canine kidney (MDCK) cells and presumably in the hypertonic renal medulla. The canine gene for the betaine gamma-amino-n-butyric acid transporter has been cloned and analyzed. The gene extends over 28 kb and consists of 18 exons. The 5' end of the gene has three alternative first exons (1A, 1B, and 1C+D). Analysis of BGT1 mRNA revealed that there is considerable divergence in the 5' untranslated sequence resulting from three different 5' end motifs (A, B, and C) followed by an alternative motif (D) as well as two internal acceptor sites for splicing. Eight kinds of BGT1 mRNA were classified into three types (A, B, and C) according to the 5' end sequence. Northern blot analysis using probes specific for the A, B, or C motif revealed that hypertonicity induces all three types in MDCK cells. Reverse transcription and polymerase chain reaction showed that each type was expressed in a tissue-specific manner. Primer extension and/or RNase protection assays as well as transfection assays into MDCK cells demonstrated that exons 1A, 1B, and 1C+D have independent transcription initiation sites under control of independent promoters. Diverse mRNA isoforms are regulated by hypertonicity and are expressed in a tissue-specific manner.