The URF13 protein, which is encoded by the maize mitochondrial T-urfl3 gene, is thought to be responsible for pathotoxin and methomyl sensitivity and male sterility. We have investigated whether T-ufl3 confers toxin sensitivity and male sterility when expressed in another plant species.The coding sequence of T-urfl3 was fused to a mitochondrial targeting presequence, placed under the control of the cauliflower mosaic virus 35S promoter, and introduced into tobacco byAgrobacterium tumefaciens-mediated transformation.Plants expressing high levels of URF13 were methomyl sensitive. Subcellular analysis indicated that URF13 is mainly associated with the mitochondria. Adding methomyl to isolated mitochondria stimulated NADH-linked respiration and uncoupled oxidative phosphorylation, indicating that URF13 was imported into the mitochondria, and conferred toxin sensitivity. Most control plants, which expressed the T-urfl3 construct lacking the mitochondrial presequence, were methomyl sensitive and contained URF13 in a membrane fraction. Subcellular fractionation by sucrose gradient centrifugation showed that URF13 sedimented at several positions, suggesting the protein is associated with various organelles, including mitochondria. No methomyl effect was observed in isolated mitochondria, however, indicating that URF13 was not imported and did not confer toxin sensitivity to the mitochondria. Thus, URF13 confers toxin sensitivity to transgenic tobacco with or without import into the mitochondria. There was no correlation between the expression of URF13 and male sterility, suggesting either that URF13 does not cause male sterility in transgenic tobacco or that URF13 is not expressed in sufficient amounts in the appropriate anther cells.