The autoinducer-2 molecule, AI-2, is considered to be a universal signal for regulating a wide variety of physiological processes in bacteria by modulating gene expression. Studies were conducted to observe how Escherichia coli cells would respond to subtherapeutic tetracycline concentrations under continuous culture (chemostat) conditions, to observe AI-2 activity within the probiotic chicken microbial consortium (Preempt® CF3; MS Bioscience, Dundee, IL) under in vitro conditions simulating a chicken cecum, and to observe the AI-2 activity in vivo within a chicken cecum as a function of exposure to subtherapeutic levels of chlortetracycline, tylosin, and vancomycin. The AI-2 activity in the E. coli continuous culture showed a 20-fold increase over baseline conditions for up to 24 hours. When the E. coli culture was subsequently exposed to pulses of chlortetracycline additions at subtherapeutic concentrations (2 μg/ml), AI-2 activity increased with increasing levels of tetracycline additions. The probiotic Preempt CF3 culture, however, did not exhibit any AI-2 activity in Viande Levure (VL) medium in the presence or absence of subtherapeutic levels of tetracycline. In vivo studies in the cecum of poultry chicks demonstrate that though AI-2 activity increased initially in the presence of vancomycin, there was no significant increase in AI-2 activity in the presence of tetracycline or tylosin. These results indicate that detectable levels of AI-2 activity are not evident within the probiotic culture (CF3) or within the chicken cecum. Understanding the relationships between AI-2 activity and microbial consortia characteristics could provide clues regarding the vulnerability of poultry chicks to enteric bacterial pathogen colonization.