This study investigated whether toxin B of Clostridium difficile (C. difficile) can activate human submucosal neurons and the pathways involved. Isolated segments of human colon were placed in organ culture for 3 h in the presence of toxin B or IL-1. Whole mounts of internal submucosal plexus were stained with antibodies against c-Fos, neuron-specific enolase (NSE), vasoactive intestinal polypeptide (VIP) and substance P (SP). The membrane potential (Vm) response of submucosal neurons to local application of toxin B and IL-1 was determined by a multisite optical recording technique. Toxin B (0.1 to 10 ng/ml) increased the proportion of c-Fos-positive neurons dose-dependently as compared to the control. In the presence of toxin B (10 ng/ml), most c-Fos-positive neurons were immunoreactive for VIP (79.8±22.5%), but only 19.4±14.0% for SP. Toxin B induced a rapid rise in IL-1 mRNA level and a six-fold increase in IL-1 protein in supernatant after 3 h of incubation. The c-Fos expression induced by toxin B was reduced dose-dependently by IL-1 receptor antagonist (0.1-10 ng/ml). Il-1 significantly increased c-Fos expression in submucosal neurons as compared to the control (34.2±10.1% vs. 5.1±1.3% of NSE neurons). Microejection of toxin B had no effect on the Vm of enteric neurons. Evidence of a direct excitatory effect of Il-1 on Vm was detected in a minority of enteric neurons. Therefore, toxin B of C. difficile activates VIP-positive submucosal neurons at least in part via an indirect IL-1-dependent pathway.