We have isolated a mouse cDNA clone encoding 3'-phosphoadenylylsulfate-galactosylceramide 3'-sulfotransferase (cerebroside sulfotransferase; CST; EC 2.8.2.11) from a kidney cDNA library, using a human CST cDNA clone [Honke, K., Tsuda, M., Hirahara, Y., Ishii, A., Makita, A. & Wada, Y. (1997) J. Biol. Chem. 272, 4864-4868] as a probe. A recombinant protein of the cloned cDNA showed CST activity. The deduced protein is composed of the same 423 amino acids as human CST and its sequence exhibits 84% identity with that of the human counterpart. Northern-blot analysis and subquantitative reverse transcription-PCR (RT-PCR) analysis showed that the CST gene is preferentially transcribed in stomach, small intestine, brain, kidney, lung, and testis, in that order. To examine differences in transcripts in various tissues, we isolated CST cDNA clones from stomach, small intestine, brain, kidney, and testis by 5'-RACE analysis. We found seven different nucleotide sequences in the 5'-UTR, while the DNA sequences of all the isolated cDNA clones were identical in the coding region. In addition, we isolated CST genomic DNA clones from a mouse genomic library. The clones covered all the 5'-UTR sequences and coding exons including 3'-UTR. RT-PCR analyses of CST mRNAs from various tissues confirmed that CST transcripts are tissue-specifically spliced by alternative use of multiple exons 1. These observations suggest that the tissue-specific expression of the CST gene is explained by alternative usage of multiple 5'-UTR exons flanked with tissue-specific promoters.