The performance of an expression system based on a fusion of the bacteriophage 434-repressor to the VP16 activation domain of Herpes simplex virus type 1 (434:VP16) was tested after stable integration into Arabidopsis. A special feature of this system was the use of the monocot maize ubiquitin1 and rice actin1 promoters to drive the expression of the 434:VP16 activator and 434-repressor, respectively. Our results demonstrate that the maize ubiquitin1 and the rice actin1 promoters, each of which contain introns, are active in Arabidopsis and can be used to express genes in this dicot species. Activation of gene expression after co-integration of the activator and reporter cassettes into the same genomic locus resulted in a higher activation level (84-fold activation) compared to crossing individual lines expressing only the activator or the operator reporter cassette alone (9-fold activation). Increasing the number of operator elements in the reporter cassette from 1 to 4 increased the activation level in cross-activated lines to an average of 281-fold with one combination of parental lines giving a 900-fold activation. Simultaneous expression of the 434-repressor protein driven by the rice actin promoter resulted in a significant decrease in the 434:VP16 mediated reporter gene expression. Nevertheless, an overall induction via 434:VP16 was possible even in the presence of the 434-repressor protein. This feature is important for genes which need to be absolutely repressed except under activating conditions. To our knowledge this investigation is the first report on the use of the 434:VP16 chimeric activator in an expression system in stably transformed plant lines.