Tonicity-responsive enhancer/osmotic response element-binding protein (TonEBP/OREBP) is a Rel protein that activates transcription of osmoprotective genes at high extracellular NaCl. Other Rel proteins NFAT1-4 and NF-kappaB complex with activator protein-1 (AP-1) to transactivate target genes through interaction at composite NFAT/NF-kappaB.AP-1 sites. TonEBP/OREBP target genes commonly have one or more conserved AP-1 binding sites near TonEBP/OREBP cognate elements (OREs). Also, TonEBP/OREBP and the AP-1 proteins c-Fos and c-Jun are all activated by high NaCl. We now find, using an ORE.AP-1 reporter from the target aldose reductase gene or the same reporter with a mutated AP-1 site, that upon stimulation by high extracellular NaCl, 1) the presence of a wild type, but not a mutated, AP-1 site contributes to TonEBP/OREBP-dependent transcription and 2) AP-1 dominant negative constructs inhibit TonEBP/OREBP-dependent transcription provided the AP-1 site is not mutated. Using supershifts and an ORE.AP-1 probe, we find c-Fos and c-Jun present in combination with TonEBP/OREBP. Also, c-Fos and c-Jun coimmunoprecipitate with TonEBP/OREBP, indicating physical association. Small interfering RNA knockdown of either c-Fos or c-Jun inhibits high NaCl-induced increase of mRNA abundance of the TonEBP/OREBP target genes AR and BGT1. Furthermore, a dominant negative AP-1 also reduces high NaCl-induced increase of TonEBP/OREBP transactivating activity. Inhibition of p38, which is known to stimulate TonEBP/OREBP transcriptional activity, reduces high NaCl-dependent transcription of an ORE.AP-1 reporter only if the AP-1 site is intact. Thus, AP-1 is part of the TonEBP/OREBP enhanceosome, and its role in high NaCl-induced activation of TonEBP/OREBP may require p38 activity.