The rat gap junction protein connexin40 (rCx40) has a characteristic developmental and regional expression pattern, for which the exact regulatory mechanisms are not known. To identify the molecular factors controlling Cx40 expression, its proximal promoter was characterized. The proximal rCx40 promoter is the most conserved noncoding region within the Cx40-gene known thus far and contains five potential binding sites for Sp-family transcription factors. The binding of both Sp1 and Sp3 to each of these DNA elements was demonstrated by EMSA. Luciferase assays of the natural rCx40 proximal promoter or mutated derivatives in Cx40-expressing (NCM, primary rat neonatal cardiomyocytes and A7r5, rat smooth muscle embryonic thoracic aorta cells) and -nonexpressing cells (N2A, mouse neuroblastoma cells) revealed that all sites are contributing to basal promoter activity. Trans-activation assays in Drosophila Schneider line 2 cells demonstrated that Sp1 and Sp3 activate the rCx40 proximal promoter in a dose-dependent and additive manner.