The M2 transmembrane peptide (M2TMP) of the influenza A virus forms a tetrameric helical bundle that acts as a proton-selective channel important in the viral life cycle. The side-chain conformation of the peptide is largely unknown and is important for elucidating the proton-conducting mechanism and the channel stability. Using a F-19 spin diffusion NMR technique called CODEX, we have measured the oligomeric states and interhelical side chain-side chain F-19-F-19 distances at several residues using singly fluorinated M2TMP bound to DMPC bilayers. F-19 CODEX data at a key residue of the proton channel, TrP4], confirm the tetrameric state of the peptide and yield a nearest-neighbor interhelical distance of similar to 11 A under both neutral and acidic pH. Since the helix orientation is precisely known from previous N-15 NMR experiments and the backbone channel diameter has a narrow allowed range, this 19F distance constrains the TrP41 side-chain conformation to t(90) (chi(1) approximate to 180 degrees, chi(2) approximate to 90 degrees). This TrP41 rotamer, combined with a previously measured N-15-C-13 distance between His(37) and TrP411, suggests that the His(37) rotamer is t-160. The implication of the proposed (His(37), Trp(41)) rotamers to the gating mechanism of the M2 proton channel is discussed. Binding of the antiviral drug amantadine to the peptide does not affect the F-F distance at TrP41. Interhelical F-19-F-19 distances are also measured at residues 27 and 38, each mutated to 4-F-19-Phe. For V27F-M2TMP, the F-19-F-19 distances suggest a mixture of dimers and tetramers, whereas the L38F-M2TMP data indicate two tetramers of different sizes, suggesting side chain conformational heterogeneity at this lipid-facing residue. This work shows that F-19 spin diffusion NMR is a valuable tool for determining long-range intermolecular distances that shed light on the mechanism of action and conformational heterogeneity of membrane protein oligomers.