The present study was designed to determine whether arginine vasopressin (AVP) stimulates NO production in the epithelial collecting duct cells of the inner medulla (IMCDs) and if this is mediated through Ca 2+ signaling. Thin tissue layers containing IMCDs were dissected from Sprague-Dawley rats. Intracellular Ca 2+ concentration ([Ca 2+ ] i ) and NO production were measured in IMCDs by a fluorescence imaging system with the use of fura 2-AM and the cell-permeable form of the NO-sensitive dye 4,5-diaminofluorescein (DAF-2), respectively. AVP (100 nmol/L) produced a rapid peak increase in [Ca 2+ ] i of 320±70 nmol/L within a few seconds and a sustained increase of 120±62 nmol/L. The peak increase in [Ca 2+ ] i was followed by a significant increase of NO production (34±7 U). This was similar to that produced by 20 μmol/L of the NO donor DETA-NONOate (42±11 U). The NO scavenger carboxy-PTIO (100 μmol/L) or depletion of [Ca 2+ ] i by preincubation with 5 μmol/L of the Ca 2+ -ATPase inhibitor thapsigargin in Ca 2+ -free buffer abolished the NO response to AVP. We conclude that AVP mobilizes Ca 2+ to produce NO in IMCDs.