AbstractPulse EPR measurements provide information on distances and distance distributions in proteins but require the incorporation of pairs of spin labels that are usually attached to engineered cysteine residues. In previous work, we demonstrated that efficient in vivo labeling of theEscherichia coliouter membrane vitamin B12transporter, BtuB, could only be achieved using strains defective in the periplasmic disulfide bond formation (Dsb) system. Here, we extend these in vivo measurements to FecA, theE. coliferric citrate transporter. As seen for BtuB, pairs of cysteines cannot be labeled when the protein is present in a standard expression strain. However, incorporating plasmids that permit an arabinose induced expression of FecA into a strain defective in the thiol disulfide oxidoreductase, DsbA, enables efficient spin‐labeling and pulse EPR of FecA in cells. A comparison of the measurements made on FecA in cells with measurements made in reconstituted phospholipid bilayers suggests that the cellular environment alters the behavior of the extracellular loops of FecA. In addition to these in situ EPR measurements, the use of a DsbA minus strain for the expression of BtuB improves the EPR signals and pulse EPR data obtained in vitro from BtuB that is labeled, purified, and reconstituted into phospholipid bilayers. The in vitro data also indicate the presence of intermolecular BtuB‐BtuB interactions, which had not previously been observed in a reconstituted bilayer system. This result suggests that in vitro EPR measurements on other outer membrane proteins would benefit from protein expression in a DsbA minus strain.