SummaryTransgenic tobacco plants expressing three different forms of Arabidopsis plant peptide deformylase (AtDEF1.1, AtDEF1.2 and AtDEF2; EC 3.5.1.88) were evaluated for resistance to actinonin, a naturally occurring peptide deformylase inhibitor. Over‐expression of either AtDEF1.2 or AtDEF2 resulted in resistance to actinonin, but over‐expression of AtDEF1.1 did not. Immunological analyses demonstrated that AtDEF1.2 and AtDEF2 enzymes were present in both stromal and thylakoid fractions in chloroplasts, but AtDEF1.1 was localized to mitochondria. The highest enzyme activity was associated with stromal AtDEF2, which was approximately 180‐fold greater than the level of endogenous activity in the host plant. Resistance to actinonin cosegregated with kanamycin resistance in Atdef1.2‐D and Atdef2‐D transgenic plants. Here, we demonstrate that the combination of plant peptide deformylase and peptide deformylase inhibitors may represent a native gene selectable marker system for chloroplast and nuclear transformation vectors, and also suggest plant peptide deformylase as a potential broad‐spectrum herbicide target.