We have used tryptophan fluorescence spectroscopy to characterize the binding affinities of an Escherichia coli LamB signal peptide family for lipid vesicles. These peptides harbor charged residue substitutions in the hydrophobic core region. Titrations of peptides with vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-3-phosphoglycerol (65:35 mol%), in conjunction with evaluation of peptide dissociation rates from these vesicles, were used to determine binding parameters quantitatively. We find that under low ionic strength conditions, point mutations introducing negatively charged aspartate residues substantially reduce peptide affinity relative to the wild-type peptide. However, the difference between wild-type and mutant peptide affinities was much lower under approximately physiological ionic strength. In addition, the lipid affinities of model surface-binding and transmembrane peptides were determined. These comparative studies with signal and model peptides permitted semi-quantitative deconvolution of signal peptide binding into electrostatic and hydrophobic components. We find that both interactions contribute significantly to binding, although the theoretically available hydrophobic free energy is largely offset by unfavorable polar-group effects. The implications of these results for understanding the potential roles of the signal sequence in protein translocation are discussed.