The effects of nitric oxide (NO) on intracellular Ca2+ concentration ([Ca2+]i) were studied in enzymatically dispersed interstitial cells (ICs) and smooth muscle cells (SMCs) isolated from canine colon. [Ca2+]i was monitored by using fluo-3 and video fluorescence imaging techniques. Exogenous NO caused an increase in [Ca2+]i in ICs and a decrease in [Ca2+]i in SMCs. Effects of NO on ICs were not blocked by removal of extracellular Ca2+ but were blocked by ryanodine, suggesting that NO caused release of Ca2+ from intracellular stores. When [Ca2+]i was elevated in an IC by micropressure ejection of Bay K 8644, [Ca2+]i decreased in nearby SMCs, suggesting release of a diffusible substance. The diffusible substance may be NO or an NO-related substance based on blockade of transmission by NG-nitro-L-arginine methyl ester, NG-monomethyl-L-arginine, or oxyhemoglobin. The elevation of [Ca2+]i in ICs by NO, which, in turn, might cause further release of NO and elevation of [Ca2+]i, suggests a positive feedback and amplification mechanism in these cells. Elevation of [Ca2+]i in SMCs had no effect on adjacent SMCs. Our data suggest that ICs may play a central role in amplification of NO signaling and propagation of inhibitory wave fronts.