To determine the molecular mechanisms by which N-cadherin transcription is regulated, we cloned and sequenced a 3681-bp of the 5'-flanking region of the human N-cadherin gene. Deletion analysis of the proximal region identified a minimal 318-bp region with strong promoter activity in human osteoblasts. The cryptic promoter is characterized by high GC content and a GA-rich binding core that may bind zing finger transcription factors. Electrophoretic mobility shift assays (EMSA), competition and supershift EMSA revealed that an Sp1/Sp3 binding site acts as a basal regulatory element of the promoter in osteoblasts. Incubation of osteoblast nuclear extracts with -163/-131 wild-type probe containing the GA-rich binding core revealed another specific complex, which was not formed with a -163/-131 probe mutated in the GA repeat. EMSA identified the nuclear factor involved as myeloid zinc finger-1 (MZF1). Mutation analysis showed that Sp1/Sp3 and MZF1 binding sites contribute to basal promoter activity. Cotransfection analyses showed that Sp1 and MZF1 overexpression increases whereas Sp3 antagonizes Sp1-induced N-cadherin promoter activity in osteoblasts. RT-PCR analysis showed that human osteoblastic cells express MZF1 and that Sp1/MZF1 overexpression increased N-cadherin expression. These results indicate that Sp1/Sp3 and MZF1 are important transcription factors regulating N-cadherin promoter activity and expression in osteoblasts.