Repeated gene targeting by recombinase-mediated cassette exchange (RMCE) is an efficient tool for the study of gene function and regulation because of the high predictability and repeatability of gene expression. We have developed the site-directed integration (SDI) vector system for Agrobacterium -mediated transformation to precisely integrate a single copy of a desired gene into a predefined chromosomal locus in the absence of any coexpressed selection marker gene (Nanto et al. Plant Biotechnol J 3:203–214, 2005; Nanto and Ebinuma Transgenic Res 17:337–344, 2008; Nanto et al. Plant Cell Rep 28:777–785, 2009; Ebinuma and Nanto (2009) Marker-free targeted transformation, in Molecular techniques in crop improvement (2nd Edition). (Jain, S. M. and Brar, D. S. eds.), Springer Netherlands, pp. 527–543; Ebinuma and Nanto in preparation). The SDI vector system consists of a target vector to introduce the target cassette and an exchange vector to reintroduce the exchange cassette for gene replacement. We describe the molecular design and experimental protocol that can efficiently enrich RMCE events through the removal of randomly integrated copies and select clean marker-free targeted transgenic plants by using a negative marker.