The Sec machinery constitutes the major pathway for protein translocation in bacteria. SecA is thought to act as a molecular motor driving translocation of the preprotein across the membrane by repeated ATP-driven cycles of insertion and retraction at the translocon channel. SecA is predominately a dimer under physiological conditions; however, its oligomeric state during active protein translocation is still unresolved. Five SecA crystal structures have been determined, each displaying a different dimer interface, suggesting that SecA may adopt different dimer configurations. In this study, a Förster resonance energy transfer approach was utilized with nine functional monocysteine SecA mutants labeled with appropriate dyes to determine the predominant solution state dimer. Three different dye pairs allowed interprotomer distances ranging from 20 to 140 Å to be investigated. Comparison of 15 experimentally determined distances with those predicted from X-ray structures showed the greatest agreement with the Bacillus subtilis SecA antiparallel dimer structure [Hunt, J., Weinkauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, D. B., and Deisenhfer, J. (2002) Science 297, 2018-2026]. The binding of two signal peptides to SecA was also examined to determine their effect on SecA dimer structure. We found that the SecA dimer is maintained upon peptide binding; however, the preprotein cross-linking domain (PPXD) and helical wing domain regions experience significant conformational changes, and the PPXD movement is greatly enhanced by binding of an extended signal peptide containing 19 additional residues. Modeling of an "open" antiparallel dimer structure suggests that binding of preprotein to SecA induces an activated open conformation suitable for binding to SecYEG.