Assembly of bioactive natural compounds through the action of nonribosomal peptide synthetases (NRPSs) relies on the specific interplay of modules and domains along these multiple mega-enzymes. As the C termini of several bacterial NRPSs often harbor epimerization (E) domains that generate D-amino acids, these seem to facilitate the ordered intermolecular enzymatic interaction and the directed transfer of intermediates. To elucidate this bifunctional role, E domains in recombinant bimodular proteins derived from the tyrocidine synthetase B were investigated. By utilizing sequent tryptic proteolysis and HPLC Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), we could directly interrogate and determine the formation of intermediates attached to the TycB(3)-PCP domain of wild-type TycB(2-3) and to the E domain exchange enzyme TycB(2-3)-ATCAT/E(tycA). In addition, the two proteins and a version of TycB(2-3) fused to the communication-mediating (COM) domain of TycA were applied in product formation assays with TycB(1) to corroborate E domain impact on intermodular NRPS interaction. Significant functional differences between the C-terminal aminoacyl- and peptidyl-E domains were observed in terms of in trans interaction and misinitiation. E domains originating from elongation modules (peptidyl-E domains) seem to be optimized for regulation of the progression of peptide bond formation, epimerization, and intermediate transfer to the downstream module, whereas E domains of initiation modules (aminoacyl-E domains) impair upstream condensation and cause misinitiation. The selection of E domains is therefore decisive for successful application in biocombinatorial engineering of nonribosomal peptides.