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We have isolated and characterized a genomic fragment encompassing the first six exons and 2.6 kbp 5' flanking sequence of the rat cystathionine beta-synthase (CBS) gene. A previously unknown exon approximately 3 kbp upstream of exon 1 was identified. The transcription start site was mapped to approximately 3 kbp upstream from the translation start codon and contains a consensus cap signal. The putative promoter region contains three GC boxes, in both orientations, and no TATA box. We have also compared a 1171-bp-long DNA sequence of the 5' end of the rat CBS gene with the homologous mouse region of 1125 bp. We found two homologous Sp1 sites in the mouse gene and an overall sequence conservation of 70% with 88-89% similarity in the 80-bp regions surrounding the intron 0 splice sites.