AbstractProline (Pro) is one of the most widely distributed osmolytes in water-stressed plants. We previously isolated from Arabidopsis a gene encoding Pro dehydrogenase (ProDH), a mitochondrial enzyme involved in the first step of the conversion of Pro to glutamic acid. The ProDH gene in Arabidopsis is up-regulated by rehydration after dehydration but is down-regulated by dehydration.ProDH is also induced by l-Pro and hypoosmolarity. The induction of ProDH expression under rehydration seems to be caused by both accumulated Pro and hypoosmolarity. We analyzed a DNA region that is located 5′ to the transcription start site (a promoter region) of ProDH to identify cis-acting elements involved in l-Pro-induced and hypoosmolarity-induced expression in transgenic tobacco (Nicotiana tabacum) and Arabidopsis plants. We found that a 9-bp sequence, ACTCATCCT, in the ProDH promoter is necessary for the efficient expression of ProDH in response to l-Pro and hypoosmolarity. Moreover, ACTCAT is a core cis-acting element, which we have called Pro- or hypoosmolarity-responsive element (PRE), that is necessary forl-Pro-responsive and hypoosmolarity-responsive expression of ProDH. Microarray and RNA gel-blot analyses showed that 21 l-Pro-inducible genes have the PRE sequences in their promoter regions. These results indicate that the PRE sequence play an important role in the l-Pro-responsive gene expression.