The coat protein of bacteriophage Pf3 is inserted into the plasma membrane of Escherichia coli by the insertase YidC. To identify which of the six transmembrane regions of YidC bind the single-spanning Pf3 coat protein during membrane protein biogenesis, we used the disulfide cross-linking approach. We generated single cysteines in each of the transmembrane regions of YidC and in the center of the hydrophobic region of Pf3 coat protein. We found that the substrate Pf3 coat contacts the first and third transmembrane segment (TM) of YidC as crosslinks between these two proteins can be formed in vivo during membrane biogenesis. A detailed disulfide-mapping study revealed that one face of TM3 of YidC makes contact with the Pf3 protein.