The role of nitric oxide (NO) in abscisic acid (ABA)-induced stomatal closure is under debate. We conducted experiments in Viciafaba leaves using NO gas as well as sodium nitroprusside (SNP), a NO-donor compound, and compared their effects to those of ABA. In epidermal strips, stomatal closure was induced by ABA, but not by NO, casting doubt on the role of NO in ABA-mediated stomatal closure. Leaf discs and intact leaves showed a dual dose response to NO: stomatal aperture widened at low, while narrowed at high dosage. Overcoming stomatal resistance by high CO2 concentration ([CO2]) restored photosynthesis in ABA-treated leaf discs, but not in those exposed to NO. NO inhibited photosynthesis immediately, causing an instantaneous increase in intercellular [CO2] (Ci), followed by stomatal closure. However, lowering Ci by using low ambient [CO2] showed that Ci was not the main factor in NO-induced stomatal closure. In intact leaves, rate of stomatal closure by NO was about one order of magnitude less than after ABA application. Because of different kinetics of photosynthesis and stomatal closure, we conclude that NO is not likely to be the key factor in ABA-induced rapid stomatal closure, but that NO fine-tunes stomatal aperture via different pathways. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.