We have previously shown that sigmoid circular muscle cells from ulcerative colitis (UC) patients exhibit reduced contraction and Ca²⁺ signaling in response to the endogenous neurotransmitter neurokinin A (NKA), and that IL-1 and H₂O₂ are elevated in UC circular muscle and may contribute to reduced contraction and impaired Ca²⁺ signaling in UC. In addition, we found that nitric oxide (NO) levels were significantly increased in UC circular muscle. To establish the site of origin for IL-1, H₂O₂ and NO, we assembled an in vitro system where normal or UC "mucosa" were sealed between two chambers filled with oxygenated Krebs' solution. Because the "mucosa" consists of full thickness mucosa and submucosa, it is expected that whatever is released into the undernatant from the submucosal side may diffuse to the circular muscle layer in the intact colon. Treatment for two hours of normal sigmoid circular muscle cells with undernatants collected from the UC submucosal side (US-S) significantly decreased contraction induced by NKA and thapsigargin, and the NKA- and caffeine-induced Ca²⁺ signal in Ca²⁺ free medium. In addition, UC mucosa released significantly more H₂O₂, IL-1 and NO into the undernatant on its submucosal side than normal mucosa. The reduction in contraction and Ca²⁺ signal induced by UC-S was partially reversed by pretreatment with an IL-1 antibody or with the H₂O₂ scavenger catalase. The NO scavenger hemoglobin partially prevented UC-S-induced reduction in contraction and Ca²⁺ signal in response to NKA, but not the reduced response to thapsigargin or caffeine. Sodium nitroprusside inhibited the NKA, but not the caffeine -induced Ca²⁺ signal. We conclude that in UC the mucosa produces IL-1, H₂O₂ and NO and that these products may contribute to the impaired Ca²⁺ release and altered sigmoid muscle contractility typically observed in UC.