Cystathionine beta-synthase (CBS) catalyzes the condensation of serine with homocysteine to form cystathionine and occupies a crucial regulatory position between the methionine cycle and transsulfuration. The human cystathionine beta-synthase gene promoters -1a and -1b are expressed in a limited number of tissues and are coordinately regulated with proliferation through a redox-sensitive mechanism. Site-directed mutagenesis, DNase I footprinting and deletion analysis of 5276 bp of 5' proximal -1b flanking sequence revealed that this region does not confer tissue-specific expression and that 210 bp of proximal sequence is sufficient for maximal promoter activity. As little as 32 bp of the -1b proximal promoter region is capable of driving transcription in HepG2 cells, and this activity is entirely dependent upon the presence of a single overlapping Sp1/Egr1 binding site. Co-transfection studies in Drosophila SL2 cells indicated that both promoters are transactivated by Sp1 and Sp3 but only the -1b promoter is subject to a site-specific synergistic regulatory interaction between Sp1 and Sp3. Sp1-deficient fibroblasts expressing both Sp3 and NF-Y were negative for CBS activity. Transfection of these cells with a mammalian Sp1 expression construct induced high levels of CBS activity indicating that Sp1 has a critical and indispensable role in the regulation of cystathionine beta-synthase. Sp1 binding to both CBS promoters is sensitive to proliferation status and is negatively regulated by Kruppel-like factors in co-transfection experiments suggesting a possible mechanism for the tissue specific regulation of cystathionine beta-synthase.