Agrobacterium tumefaciens pTiA6 virE promoter has a vir box, an inverted repeat (IR) sequence, a putative -35 region and a consensus -10 region. To study how the IR sequence of the virE promoter plays a role in virE gene expression, various mutants were constructed by base substitution and deletion in the virE promoter region. Substitution of the 3'-end region of the IR sequence, 5'-TCCGTTTCAA-3' to 5'-GCGGCCGCTC-3' displayed 2.6% of the native virE promoter activity. A deletion mutant of 5'-CGTTTCAA-3' on the 3'-end region of the IR sequence expressed 6% of the native virE promoter activity. These mutational analyses demonstrated that the IR sequence of the virE promoter plays a role as a cis-acting element in virE expression.