Parallel experimental measurements and theoretical calculations have been used to investigate the energetics of electrostatic interactions in the complex formed between a 22 residue, alpha-helical peptide from the N protein of phage lambda and its cognate 19 nucleotide box B RNA hairpin. Salt-dependent free energies were measured for both peptide folding from coil to helix and peptide binding to RNA, and from these the salt-dependence of binding pre-folded, helical peptide to RNA was determined ( partial differential (DeltaG degrees (dock))/ partial differential log[KCl]=5.98(+/-0.21)kcal/mol). (A folding transition taking place in the RNA hairpin loop was shown to have a negligible dependence on salt concentration.) The non-linear Poisson-Boltzmann equation was used to calculate the same salt dependence of the binding free energy as 5.87(+/-0.22)kcal/mol, in excellent agreement with the measured value. Close agreement between experimental measurements and calculations was also obtained for two variant peptides in which either a basic or acidic residue was replaced with an uncharged residue, and for an RNA variant with a deletion of a single loop nucleotide. The calculations suggest that the strength of electrostatic interactions between a peptide residue and RNA varies considerably with environment, but that all 12 positive and negative N peptide charges contribute significantly to the electrostatic free energy of RNA binding, even at distances up to 11A from backbone phosphate groups. Calculations also show that the net release of ions that accompanies complex formation originates from rearrangements of both peptide and RNA ion atmospheres, and includes accumulation of ions in some regions of the complex as well as displacement of cations and anions from the ion atmospheres of the RNA and peptide, respectively.