The small hydrophobic (SH) protein of human respiratory syncytial virus (HRSV) has been efficiently expressed in Escherichia coli. In analogy to small hydrophobic proteins encoded by other RNA viruses, membrane permeability changes to low-molecular-weight compounds were detected in bacteria expressing HRSV SH protein. These changes implied, at least, the entry of both the protein synthesis inhibitor hygromycin B and the beta-galactoside substrate o-nitrophenyl-beta-d-galactopyranoside and the exit of preloaded [3H]uridine from bacterial cells. Site-directed mutagenesis indicated that the C-terminal end of SH is needed for induction of membrane permeability changes. In addition, amino acid substitution at residue 32 (Ile to Lys) abolished that activity. This was correlated with a drastic increase in SH electrophoretic mobility and a decrease of the predicted values of alpha-helix for all residues of the SH transmembrane domain. Other sequence changes have either partial effect or no effect on the membrane permeability changes induced by the SH protein. However, none of the mutations abrogated the association of SH protein with bacterial membranes, indicating that incorporation of SH protein to membranes is not sufficient to induce the observed changes. Membrane permeability changes then might provide a useful test for the identification of key amino acid residues in this unique HRSV gene product.