By means of in vivo footprinting, we examined the putative cis-acting DNA elements located between -50 and -122 of rat Na+/K(+)-ATPase alpha 1 subunit gene ATP1A1. Proximal and distal GC box sequences and a consensus sequence for the active transcription factor (ATF) were protected for all the tissues examined (kidney, brain and liver). Putative cooperation between two binding factors on the ATF site and the proximal GC box was observed. The overall in vivo footprinting profiles of the three tissues did not exhibit any marked differences that could account for the variation in the extent of tissue-specific transcription. The alpha 1 regulatory element (ARE) found by Suzuki-Yagawa et al. does not appear to be an element responsible for tissue-specific regulation of the gene.