The structure of the putative feline immunodeficiency virus (FIV) ribosomal frameshifting pseudoknot (PK) has been investigated by a mass spectrometric three-dimensional (MS3D) approach, which involves the application of established solvent-accessibility probes and chemical crosslinkers with detection by electrospray ionization (ESI) Fourier transform mass spectrometry (FTMS). Regardless of their size, probed substrates can be treated with ribonucleases and analyzed by ESI-FTMS to obtain the correct position of chemically modified nucleotides. Protection maps and distance information can be utilized to generate 3D models using the constraint satisfaction algorithm provided by MC-SYM and the energy minimization modules included in CNS. Control experiments were performed on a mutant of mouse mammary tumor virus pseudoknot (VPK), for which an NMR structure is available. Comparison between the MS3D model and the high-resolution structure provided a ∼3 Å root-mean-square deviation calculated from all the atoms present in double-stranded regions. Applied to FIV-PK, the MS3D approach confirmed that the selected sequence could fold into an actual pseudoknot, supporting the sequence alignment predictions. Characteristic features of H-type pseudoknots were recognized immediately, but a putative A13–U30 pair was not observed at the stem junction, making FIV-PK resemble VPK more closely than the initially suggested simian retrovirus type-1 pseudoknot. In our model, the unpaired U30 protrudes into the medium, while the hinging A13 assumes a stacked conformation that enables the stems to form a ∼60° bend and relieve the strain caused by a short loop 1. The model provided the basis to explain the different alkylation patterns observed in the absence and presence of Mg 2+, suggesting the possible formation of a specific metal-binding site between loop 1 and stem 2. This instance illustrates how the MS3D model of FIV-PK can be utilized effectively to generate hypotheses and support functional observations in the absence of a high-resolution structure.