ABSTRACT: Botulinum toxin (BoNT) is a potent neurotoxin produced by toxigenic strains of Clostridium botulinum. Botulinum toxin poses a major threat since it could be employed in a deliberate attack on the U.S. food supply. Furthermore, BoNT may be liberated in any insufficiently processed food containing a reduced oxygen atmosphere. Hence, rapid and reliable detection of BoNT in foods is necessary to reduce risks posed through food contamination. We present a BoNT biosensor employing living neural cultures grown in vitro on microelectrode arrays (MEAs). An MEA is a culture dish with a grid of electrodes embedded in its surface, enabling extracellular recording of action potentials of neural cultures grown over the array. Pharmaceutical grade BoNT A was applied to the media bath of mature cortical networks cultured on MEAs. Both spontaneous and evoked activities were monitored over 1 wk to quantify changes in the neural population produced by BoNT A. Introduction of BoNT A resulted in an increased duration and number of spikes in spontaneous and evoked bursts relative to control cultures. Increases were significant within 48 h of BoNT A dosage (P < 0.05). Application of BoNT A also induced unique oscillatory behavior within each burst that is reminiscent of early developmental activity patterns rather than the mature cultures used here. Three or more activity peaks were observed in 50% of the BoNT dosed cultures. Control cultures exhibited only a single activity peak. Thus activity of these cortical networks measured with MEAs could provide a valuable substrate for BoNT detection.