N-terminal presequences from cDNAs encoding mitochondrion- or chloroplast-specific proteins are able, with variable efficiencies, to target preproteins to their respective organelles. In the few cases studied in which a nuclear-encoded protein is found in both these organelles, each compartment-specific isoform is encoded by a separate gene. Glutathione reductase (GR) from peas is encoded by a single nuclear gene and yet GR is distributed between chloroplasts, mitochondria and the cytosol. Previous sequence analysis of a full-length GR cDNA revealed the presence of a putative plastid transit peptide. However, expression of this cDNA in transgenic tobacco resulted in substantially elevated GR activities in both chloroplasts and mitochondria in four independent lines examined. There was no effect on expression of the endogenous tobacco GR genes. Replacement of the GR presequence with presequences from pea rbcS (chloroplast) and Nicotiana plumbaginifolia Mn-SOD (mitochondrion) resulted in targeting of GR only into the appropriate organelle. Expression of a fusion protein between the amino terminal region of GR and phosphinothricin acetyl transferase resulted in targeting of the foreign protein to chloroplasts and mitochondria. Thus, the pea GR presequence is capable of co-targeting this enzyme or a foreign protein to chloroplasts and mitochondria in vivo. This is the first example of co-targeting by a higher plant preprotein.