Pea glutathione reductase (GR) is dually targeted to mitochondria and chloroplasts by means of an N-terminal signal peptide of 60 amino acid residues. After import, the signal peptide is cleaved off by the mitochondrial processing peptidase (MPP) in mitochondria and by the stromal processing peptidase (SPP) in chloroplasts. Here, we have investigated determinants for processing of the dual targeting signal peptide of GR by MPP and SPP to examine if there is separate or universal information recognised by both processing peptidases. Removal of 30 N-terminal amino acid residues of the signal peptide (GRDelta1-30) greatly stimulated processing activity by both MPP and SPP, whereas constructs with a deletion of an additional ten amino acid residues (GRDelta1-40) and deletion of 22 amino acid residues in the middle of the GR signal sequence (GRDelta30-52) could be cleaved by SPP but not by MPP. Numerous single mutations of amino acid residues in proximity of the cleavage site did not affect processing by SPP, whereas mutations within two amino acid residues on either side of the processing site had inhibitory effect on processing by MPP with a nearly complete inhibition for mutations at position -1. Mutation of positively charged residues in the C-terminal half of the GR targeting peptide inhibited processing by MPP but not by SPP. An inhibitory effect on SPP was detected only when double and triple mutations were introduced upstream of the cleavage site. These results indicate that: (i) recognition of processing site on a dual targeted GR precursor differs between MPP and SPP; (ii) the GR targeting signal has similar determinants for processing by MPP as signals targeting only to mitochondria; and (iii) processing by SPP shows a low level of sensitivity to single mutations on targeting peptide and likely involves recognition of the physiochemical properties of the sequence in the vicinity of cleavage rather than a requirement for specific amino acid residues.