E. coli strains that contain the secY40 mutation are cold-sensitive, but protein export defects have not been observed even at the nonpermissive temperature. Here we describe experiments designed to explain the conditional phenotype associated with this allele. We found that combining the secY40 mutation with defects in the signal recognition particle targeting pathway led to synthetic lethality. Since the signal recognition particle is required for the insertion of inner membrane proteins (IMPs) into the cytoplasmic membrane but not for protein export, this observation prompted us to examine the effect of the secY40 mutation on IMP biogenesis. The membrane insertion of all IMPs that we tested was impaired at both permissive and nonpermissive temperatures in secY40 cells grown in either rich or minimal medium. The magnitude of the insertion defects was greatest in cells grown at low temperature in rich medium, conditions in which the growth defect was most pronounced. Consistent with previous reports, we could not detect protein export defects in secY40 cells grown in minimal medium. Upon growth in rich medium, only slight protein export defects were observed. Taken together, these results suggest that the impairment of IMP insertion causes the cold sensitivity of secY40 strains. Furthermore, these results provide the first evidence that the protein export and membrane protein insertion functions of the translocon are genetically separable.