Background and aims After a series of seminal works during the last decade of the 20th century nitric oxide (NO) is now firmly placed in the pantheon of plant signals. NO acts in plant-microbe interactions, responses to abiotic stress, stomatal regulation and a range of developmental processes. By considering the recent advances in plant NO biology, this review will highlight certain key aspects that require further attention. Scope and conclusions The following questions will be considered. Whilst cytosolic nitrate reductase is an important source of NO, the contributions of other mechanisms, including a poorly defined arginine oxidizing activity, needs to be characterized at molecular level. Other oxidative pathways utilising polyamine and hydroxylamine also need further attention. NO action is dependent on its concentration and spatial generation patterns. However, no single technology currently available is able to provide accurate in planta measurements of spatio-temporal patterns of NO production. It is also the case that pharmaceutical NO donors are used in studies, sometimes with little consideration of the kinetics of NO production. We here include in planta assessments of NO production from DEANO (diethylamine nitric oxide), S-nitrosoglutathione (GSNO) and sodium nitroprusside (SNP) following infiltration of tobacco leaves which could aid workers in their experiments. Further, based on current data it is difficult to define a bespoke plant NO signalling pathway, but rather NO appears to act as a modifier of other signalling pathways. Thus, early reports the NO signalling involves cGMP -as in animal systems – require revisiting. Finally, as plants are exposed to NO from a number of external sources investigations into the control of NO scavenging by such as non-symbiotic haemoglobins and other sinks for NO should feature more highly. By crystallising these questions the authors encourage their resolution through the concerted efforts of the plant NO community.