The influenza B virus BM2 proton-selective ion channel is essential for virus uncoating, a process that occurs in the acidic environment of the endosome. The BM2 channel causes acidification of the interior of the virus particle, which results in dissociation of the viral membrane protein from the ribonucleoprotein core. The BM2 protein is similar to A/M2 protein ion channel of influenza A virus (A/M2) in that it contains a HXXXW motif. Unlike the A/M2 protein, the BM2 protein is not inhibited by the antiviral drug amantadine. We used mutagenesis to ascertain the pore-lining residues of the BM2 ion channel. The specific activity (relative to wild type), reversal voltage, and susceptibility to modification by MTSEA and NEM of the mutant proteins were measured in oocytes. It was found that mutation of transmembrane (TM) domain residues Ser9, Ser12, Phe13, Ser16, His19, and Trp23 to cysteine were most disruptive for ion channel function. These cysteine mutants were also most susceptible to MTSEA and NEM modification. Furthermore, considerable amounts of dimer were formed in the absence of oxidative reagents when cysteine was introduced at positions Ser9, Ser12, Ser16, or Trp23. Based on these experimental data, a BM2 TM domain model is proposed. The presence of polar residues in the pore is a probable explanation for the amantadine-insensitivity of the BM2 protein and suggests that related, but more polar, compounds might serve as useful inhibitors of the protein.