The influenza B virus BM2 protein contains 109 amino acid residues and it is translated from a bicistronic mRNA in an open reading frame that is +2 nucleotides with respect to the matrix (M1) protein. The amino acid sequence of BM2 contains a hydrophobic region (residues 7-25) that could act as a transmembrane (TM) anchor. Analysis of properties of the BM2 protein, including detergent solubility, insolubility in alkali pH 11, flotation in membrane fractions, and epitope-tagging immunocytochemistry, indicates BM2 protein is the fourth integral membrane protein encoded by influenza B virus in addition to hemagglutinin (HA), neuraminidase (NA), and the NB glycoprotein. Biochemical analysis indicates that the BM2 protein adopts an N(out)C(in) orientation in membranes and fluorescence microscopy indicates BM2 is expressed at the cell surface. As the BM2 protein possesses only a single hydrophobic domain and lacks a cleavable signal sequence, it is another example of a Type III integral membrane protein, in addition to M(2), NB, and CM2 proteins of influenza A, B, and C viruses, respectively. Chemical cross-linking studies indicate that the BM2 protein is oligomeric, most likely a tetramer. Comparison of the amino acid sequence of the TM domain of the BM2 protein with the sequence of the TM domain of the proton-selective ion channel M(2) protein of influenza A virus is intriguing as M(2) protein residues critical for ion selectivity/activation and channel gating (H(37) and W(41), respectively) are found at the same relative position and spacing in the BM2 protein (H(19) and W(23)).