Nopaline strains of Agrobacterium tumefaciens contain a gene, tzs, that encodes a cytokinin biosynthetic prenyl transferase. The gene is located adjacent to the Ti plasmid virulence region and is constitutively expressed at low levels. As a result, bacteria containing tzs secrete low levels of zeatin into the medium. We find zeatin secretion to be induced more than 100-fold by acetosyringone, one of a number of naturally occurring phenolics produced by plants in response to wounding. Induction was very sensitive to the pH of the medium (optimum pH 5.5) and was due to massive overexpression of tzs-encoded cytokinin prenyl transferase activity. The relative ability of members of a set of phenols to induce tzs expression was examined and found to be parallel to that reported for activation of other virulence genes. A series of molecular cloning experiments established that virA and virG, two genes known to be essential to the virulence induction process, were necessary and sufficient for phenolic-induced tzs expression. Sequences present in the promoter region of tzs were found to be similar to those present in genes regulated by bacterial two-component positive regulatory systems.