Bacterial proteins destined for export from the cytoplasm often contain an N-terminal signal peptide. This short (20-30 amino acids) sequence is cleaved by a signal peptidase upon translocation to the periplasm. The sequence requirements for signal peptides have been extensively studied. Less well understood are sequence requirements on the mature protein side that are important for signal peptide cleavage. A recent finding reported a statistical bias against aromatic residues in the second position after the cleavage site (P2’) in the mature region of signal peptidase 1(LepB)-dependent secreted proteins. To determine the reason for this bias, the P2’ residue was changed to each of the three possible aromatic amino acids in maltose binding protein (MBP); a key model system of protein secretion. The wild-type and aromatic variants were purified and underwent protein staining and Western analysis to see if an increase in the precursor protein could be observed. In comparison to the wild-type, the aromatic P2’ MBP variants showed greater precursor accumulation on the protein stains. Surface Plasmon Resonance (SPR) using synthetic peptides encompassing the cleavage site region was used to assess the impact of these mutations on recognition by LepB. SPR analysis showed the P2’ aromatic species had similar on-rates, but slower off-rates in comparison to the wild-type. This decreased off-rate was also shown to be able to inhibit the binding of the wild-type peptide to LepB. Therefore, aromatic amino acids present at P2’ have an effect on the processing ability of signal peptidase 1. Mature protein residues play a role in signal peptidase processing, and their incorporation in signal sequence prediction software will improve bioinformatics analysis of bacterial secretomes.