The formation of noncovalent complexes between the HIV-1 nucleocapsid protein p7 (NC) and RNA hairpins SL2-SL4 of the Psi-recognition element was investigated by direct infusion electrospray ionization-Fourier transform mass spectrometry (ESI-FTMS). The high resolution afforded by this method provided the unambiguous characterization of the stoichiometry and composition of complexes formed by multiple equilibria in solution. For each hairpin, the formation of a 1:1 complex was found to be the primary binding mode in solutions of intermediate salt content (150 mM ammonium acetate). Binding of multiple units of NC was observed with lower affinity and a maximum stoichiometry matching the limit calculated from the number of nucleotides in the construct and the size of the footprint of NC onto single-stranded nucleic acids, thus implying the defolding of the hairpin three-dimensional (3D) structure. Dissociation constants of 62 +/- 22 nM, 178 +/- 64 nM, and 1.3 +/- 0.5 microM were determined for SL2, SL3-2, and SL4, respectively, which are similar to values obtained by spectroscopic and calorimetric methods with the additional confidence offered by a direct, rather than inferred, knowledge of the binding stoichiometry. Competitive binding experiments carried out in solutions of intermediate ionic strength, which has the effect of weakening the electrostatic interactions in solution, provided a direct way of evaluating the stabilizing contributions of H-bonding and hydrophobic interactions that are more sensitive to the sequence and structural context of the different hairpins. The relative scale of binding affinity obtained in this environment reflects the combination of contributions provided by the different structures of both the tetraloop and the double-stranded stem. The importance of the stem 3D structure in modulating the binding activity was tested by a competitive binding experiment that included the SL3-2 RNA construct, a DNA analogue of SL3 (SL3(DNA)), and a DNA analogue in which all four loop bases were replaced with abasic nucleotides (SL3(abasic)). NC was found to bind the A-type double-stranded stem of SL3-2 RNA at least 30 times more tightly than the B-type helical structure of SL3(DNA). Eliminating the stabilization provided by the interactions with the tetraloop bases made the binding of SL3(abasic) approximately 50 times weaker than that of SL3(DNA).