Exudates of dicotyledonous plants contain specific phenolic signal molecules, such as acetosyringone, which serve as potent inducers for the expression of the virulence (vir) regulon of the phytopathogen Agrobacterium tumefaciens. This induction activates the Agrobacterium T-DNA transfer process to initiate the genetic transformation of target plant cells. Wounded and metabolically active plant cells are particularly susceptible to Agrobacterium infection, and these cells specifically produce vir-inducing molecules. Most monocotyledonous, as opposed to dicotyledonous, species are resistant to Agrobacterium transformation. One hypothesis for this resistance is that nonsusceptible monocotyledonous cells fail to produce vir signal molecules and, thus, are not recognized by Agrobacterium as transformation targets. Here we demonstrate that monocotyledonous cells make such molecules, and, furthermore, we purify the inducer produced by a Triticum monococcum suspension culture that is resistant to Agrobacterium infection. This molecule is shown to correspond to ethyl ferulate [C12H14O4; 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid ethyl ester], to be more active for vir induction at low concentrations than acetosyringone, and to be produced in quantities giving significant levels of induction. Thus, at least for the wheat cell line used in this study, monocotyledonous resistance to Agrobacterium transformation must result from a block to a step of the T-DNA transfer process subsequent to vir induction.