Cystathionine beta-synthase (CBS) catalyzes the condensation of serine and homocysteine to form cystathionine, an intermediate step in the synthesis of cysteine. We previously characterized the CBS -1b minimal promoter (-3792 to -3667) and found that Sp1/Sp3, nuclear factor Y, and USF-1 were involved in the regulation of basal promoter activity (Ge, Y., Konrad, M. A., Matherly, L. H., Taub, J. W. (2001) Biochem. J. 357, 97-105). In this study, the critical cis-elements and transcription factors in the CBS -1b upstream region (-4046 to -3792) were examined in HT1080 and HepG2 cells, which differ approximately 10-fold in levels of CBS transcripts transcribed from the CBS -1b promoter. In DNase I footprint and gel shift analyses and transient transfections of mutant CBS -1b promoter constructs into HT1080 and HepG2 cells, transcriptionally important roles for Sp1/Sp3 binding to three GC boxes and one GT box and for binding of myeloid zinc finger 1-like proteins to two myeloid zinc finger 1 elements were indicated. In gel shift assays, very low levels of Sp1/Sp3 DNA-protein complexes were detected in HT1080 cells compared with HepG2 cells despite comparable levels of nuclear factor Y and USF-1 binding and similar levels of Sp1 and Sp3 proteins on Western blots. Mixing of HT1080 and HepG2 nuclear extracts resulted in no difference in total Sp factor binding in gel shift assays, thus excluding a role for an unknown activator or inhibitor in the disparate Sp1/Sp3 binding between the lines. Increased Sp1/Sp3 binding in gel shift assays was observed upon treatment of HT1080 nuclear extracts with protein kinase A, and decreased Sp1/Sp3 binding resulted from treatment of HepG2 nuclear extracts with calf alkaline phosphatase, suggesting a role for changes in Sp1/Sp3 phosphorylation in transcription factor binding and transactivation of the CBS -1b promoter. Characterization of CBS promoter structure and function should clarify the molecular bases for variations in CBS gene expression in genetic diseases and the relationship between CBS and Down syndrome.