The accessory Sec systems of streptococci and staphylococci mediate the transport of a family of large, serine-rich glycoproteins to the bacterial cell surface. These systems are comprised of SecA2, SecY2, and three core accessory Sec proteins (Asp1-3). In Streptococcus gordonii, transport of the serine-rich glycoprotein GspB requires both a unique 90-residue N-terminal signal peptide and an adjacent 24-residue segment (the AST domain). We used in vivo site-specific photo-cross-linking to identify proteins that interact with the AST domain during transport. To facilitate this analysis, the entire accessory Sec system of S. gordonii was expressed in Escherichia coli. The determinants of GspB trafficking to the accessory Sec system in E. coli matched those in S. gordonii, establishing the validity of this approach. When the photo-cross-linker was placed within the AST domain, the preprotein was found to cross-link to SecA2. Importantly, no cross-linking to SecA was detected. Cross-linking of the N-terminal end of the AST domain to SecA2 occurred regardless of whether Asp1-3 were present. However, cross-linking to the C-terminal end was dependent on the Asps. The combined results indicate that full engagement of the AST domain by SecA2 is modulated by one or more of the Asps, and suggest that this process is important for initiating transport.