Ornithine deacetylase (argE) gene as a negative selection marker gene was successfully co-transformed with SOD-APX or NDPK2 as a target gene to develop marker free transgenic rice. E.coli argE gene encodes an enzyme which activates N-acetyl phosphinothricin (N-AcPt) through deacetylation. The enzyme was reported that deacetylate N-AcPt, a non cyto-toxic compounds, to produce cyto-toxic PPT. So the argE gene can be used as a negative selection marker and N-AcPt can be used as a substrate of argE gene. Using former purification method (Kriete G., et al., 1996) was very expensive because the small amount of N-AcPt produced by the method. We developed a strategy to produce N-AcPt by means of chemical method. N-AcPt function was identified using hygromycin resistant T1 seeds on MS basal medium contained N-AcPt prior to utilization of argE gene as a negative selection marker in marker free transgenic rice. Negative selection effects were performed with T1 seeds containing argE gene under N-AcPt. This system provides a efficient negative selection effect from transgenic rice and that will be efficiently used for the production of marker gene free rice plants.