ABSTRACT The use of mixtures of microorganisms, or microbial consortia, has the potential to improve the productivity and efficiency of increasingly complex bioprocesses. However, the use of microbial consortia has been limited by our ability to control and coordinate the behaviors of microorganisms in synthetic communities. Synthetic biologists have previously engineered cell–cell communication systems that employ machinery from bacterial quorum‐sensing (QS) networks to enable population‐level control of gene expression. However, additional communication systems, such as those that enable communication between different species of bacteria, are needed to enable the use of diverse species in microbial consortia for bioprocessing. Here, we use the agr QS system from Staphylococcus aureus to generate an orthogonal synthetic communication system between Gram‐negative Escherichia coli and Gram‐positive Bacillus megaterium that is based on the production and recognition of autoinducing peptides (AIPs). We describe the construction and characterization of two types of B. megaterium “receiver” cells, capable of AIP‐dependent gene expression in response to AIPs that differ by a single amino acid. Further, we observed interspecies communication when these receiver cells were co‐cultured with AIP‐producing E. coli. We show that the two AIP‐based systems exhibit differences in sensitivity and specificity that may be advantageous in tuning communication‐dependent networks in synthetic consortia. These peptide‐based communication systems will enable the coordination of gene expression, metabolic pathways and growth between diverse microbial species, and represent a key step towards the use of microbial consortia in bioprocessing and biomanufacturing. Biotechnol. Bioeng. 2013;110: 3003–3012. © 2013 Wiley Periodicals, Inc. Synthetic interspecies communication between Gram‐negative Escherichia coli and Gram‐positive Bacillus megaterium was established using components from the Staphylococcus aureus agr quorum‐sensing system. Here, Marchand and Collins show that this communication system, which uses a novel peptide signal molecule, allows E. coli to control B. megaterium gene expression during co‐culture.