Nitric oxide (NO) has been intensively studied to elucidate the role of this enigmatic signaling molecule in plant development, metabolism and disease responses. Many studies using pharmacological and biochemical tools have demonstrated that NO functions in hormone responses, programmed cell death, defense gene induction and signal transduction pathways. NO originates from two sources in plants: nitrite and arginine. Recent studies using mutants and transgenic plants have confirmed these key findings and have gone further to identify (i) a new mechanism to modulate NO bioactivity involving hemoglobin, (ii) a gene involved in arginine-dependent NO synthesis, and (iii) a novel function for NO signaling in flowering. These findings continue to elucidate the expanding role of NO in plant biology.