A critical event in protein translocation across the endoplasmic reticulum is the structural transition between the closed and open conformations of Sec61, the eukaryotic translocation channel. Channel opening allows signal sequence insertion into a gap between the N- and C-terminal halves of Sec61. We have identified a gating motif that regulates the transition between the closed and open channel conformations. Polar amino acid substitutions in the gating motif cause a gain-of-function phenotype that permits translocation of precursors with marginally hydrophobic signal sequences. In contrast, hydrophobic substitutions at certain residues in the gating motif cause a protein translocation defect. We conclude that the gating motif establishes the hydrophobicity threshold for functional insertion of a signal sequence into the Sec61 complex, thereby allowing the wild-type translocation channel to discriminate between authentic signal sequences and the less hydrophobic amino acid segments in cytosolic proteins. Bioinformatic analysis indicates that the gating motif is conserved between eubacterial and archaebacterial SecY and eukaryotic Sec61.