AtNUDT5 is a cytosol Nudix that catalyzes the hydrolysis of a variety of substrates. In this report, a 1,387-bp 5'-flanking region of the AtNUDT5 gene was isolated from Arabidopsis thaliana. The tissue-specific activity of the 5'-flanking region was investigated by using the GUS gene as a reporter in transgenic A. thaliana plants. Weak GUS activity appeared in vascular tissues of young plants, strong GUS activity appeared in the axial roots, but no GUS activity was observed in the root cap, lateral roots, rosette leaf, mature silique and reproductive tissues such as stamen, pistil, and petal. Furthermore, by using these transgenic A. thaliana plants, results of the histochemical staining and fluorometric assays of GUS activity showed that the AtNUDT5 promoter can be activated by both avirulent Pst avrRpm1 and virulent Pst strains at 5 h post-infiltration and that the activity of AtNUDT5 promoter increased significantly at 24 h post-infiltration. Taken together, our results demonstrated that the AtNUDT5 promoter is pathogen-responsive. The promoter may be used to develop transgenic plants with an increased tolerance to pathogenic stresses.