Tescalcin, an EF-hand calcium binding protein that regulates the Na(+)/H(+) exchanger 1 (NHE1), is highly expressed in various mouse tissues such as heart and brain. Despite its potentially important role in cell physiology, the mechanisms that regulate tescalcin gene (Tesc) expression are unknown. In this study, we report two new Tesc mRNA variants (V2 and V3) and characterize the mouse Tesc promoter. The V2 and V3 transcripts result from alternative splicing of intron 5. Our results show that Tesc mRNA variants are expressed in various mouse tissues. Primer extension analysis located the transcription start site at 94 nucleotides upstream of the translation start codon. The DNA nucleotide sequence of the 5'-flanking region contained a CpG island spanning the promoter region from nucleotides -372 to +814, a canonical TATA box (-38/-32), and putative transcription factor binding sites for Sp1, EGR1, ZBP-89, KLF3, MZF1, AP2, ZF5, and CDF-1. Transient transfection of the Y1 and msc-1 cell lines with a series of 5'-deleted promoter constructs indicated that the minimal promoter region was between nucleotides -130 and -40. Electrophoresis mobility shift assays, supershift assays, and mutation studies demonstrated that Sp1 and Sp3 bind to the GC-rich motifs, a CACCC box and three GC boxes, located within the Tesc proximal promoter. Nonetheless, mutations that abolished interaction of Sp1 and Sp3 with the GC-rich motifs located within the minimal promoter region did not abrogate promoter activity in Y1 cells. Mithramycin A, an inhibitor of Sp1-DNA interaction, reduced Tesc promoter activity in msc-1 cells in a dose-dependent manner. Sp3 was a weaker transactivator compared to Sp1 in Drosophila D.mel-2 cells. However, when Sp1 and Sp3 were coexpressed, they transactivated the Tesc promoter in a synergistic manner. In Y1 cells, mutation analysis of a putative ZF5 motif located within the Tesc minimal promoter indicated that this motif was critical for activity of Tesc promoter. Taken together, the data demonstrated that Sp1 and Sp3 transcription factors cooperate positively in the regulation of Tesc promoter, and that the putative ZF5 motif is critical for its activation.