Although nitric oxide (NO) and reactive oxygen species (ROS) are essential signalling molecules required for mediation of abscisic acid (ABA)-induced stomatal closure, it is not known whether these molecules also mediate the ABA inhibition of stomatal opening. In this study, we investigated the role of NO and ROS in the ABA inhibition of stomatal opening in Vicia faba. ABA induced both NO and ROS synthesis, and the NO scavenger reduced the ABA inhibition of stomatal opening. Exogenous NO and hydrogen peroxide (H2O2) also inhibited stomatal opening, indicating that NO and ROS are involved in the inhibition signalling process. An inhibitor of nitric oxide synthase (NOS) reversed the ABA inhibition of stomatal opening. Either the NO scavenger or the NOS inhibitor also reversed the process in the H2O2 inhibition of stomatal opening. We found that in the ABA inhibition of stomatal opening, NO is downstream of ROS in the signalling process, and NO is synthesized by a NOS-like enzyme.