Three factors that bind to the positive regulatory region (ARE) of the Na+,K(+)-ATPase alpha 1-subunit gene were shown to be present in growing BALB/c-3T3 cells as shown by the gel retardation assay pattern in which three specific complexes (C1, C2 and C3) were identified. The complexes are similar to those observed in MDCK cell nuclear extracts in which linker substitution mutations in the competitor gave parallel specific effects in both cells. During the process of the cell growth cycle, the relative mobility of C3 was altered, and the amount of C1 decreased in the G0 state. All three complexes (C1, C2 and C3) disappeared and other specific complexes with higher mobilities were alternatively observed at 6 h after serum stimulation and thereafter. The expression of the mRNA for the alpha 1-subunit gene was repressed at G0 and gradually increased after serum stimulation. These results suggest that different sets of factors are responsible for the transcription of the gene at different stages of the cell cycle.