A 6-bp sequence, ACTCAT, acts as a cis-acting element involved in hypoosmolarity- and proline-responsive expression of an Arabidopsis proline dehydrogenase (ProDH) gene. Search of the database for plant cis-acting elements revealed that the ACTCAT sequence is similar to the GCN4 motif [ATGA(C/G)TCAT] that is recognized by bZIP transcription factors. To identify transcription factor(s) for regulation of ProDH, we examined whether Arabidopsis bZIPs function as transcription factors for the ACTCAT sequence. Transient expression analysis revealed that the four proteins in Group S bZIPs, AtbZIP11/ATB2, AtbZIP44, AtbZIP2/GBF5 and AtbZIP53, formed an ATB2 subgroup that activated expression of the GUS reporter gene driven by the ACTCAT sequence while other bZIPs and different families of plant transcription factors did not. The transactivation activity of the ATB2 subgroup was enhanced in a hypoosmotic condition. In a gel mobility shift assay, the recombinant proteins of the ATB2 subgroup specifically bound to the ACTCAT sequence. RNA gel blot analysis indicated that the expression of AtbZIP2/GBF5 and AtbZIP53, as well as that of ProDH, is induced by hypoosmolarity. Moreover, we showed that the sGFP::AtbZIP11/ATB2 fusion protein is localized in the nucleus. These results suggest that the ATB2 subgroup functions as a transcriptional activator for hypoosmolarity-inducible ProDH in Arabidopsis: