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Over the past 20 years, nitric oxide (NO) research has generated a lot of interest in various aspects of plant biology. It is now clear that NO plays a role in a wide range of physiological processes in plants. However, in spite of the significant progress that has been made in understanding NO biosynthesis and signaling in planta, several crucial questions remain unanswered. Here we highlight several challenges in NO plant research by summarizing the latest knowledge of NO synthesis and by focusing on the potential NO source(s) and players involved. Our goal is also to provide an overview of how our understanding of NO signaling has been enhanced by the identification of array of genes and proteins regulated by NO.