Most bacterial exported proteins cross the cytoplasmic membrane as unfolded polypeptides. However, little is known about how they fold during or after this process due to the difficulty in detecting folding intermediates. Here we identify cotranslational and posttranslational folding intermediates of a periplasmic protein in which the protein and DsbA, a periplasmic disulfide bond-forming enzyme, are covalently linked by a disulfide bond. The cotranslational mixed-disulfide intermediate is, upon further chain elongation, resolved, releasing the oxidized polypeptide, thus allowing us to follow the folding process. This analysis reveals that two cysteines that are joined to form a structural disulfide can play different roles during the folding reaction and that the mode of translocation (cotranslational verse posttranslational) can affect the folding process of a protein in the periplasm. The latter finding leads us to propose that the activity of the ribosome (translation) can modulate protein folding even in an extracytosolic compartment.