Nitric oxide (NO) is a reactive gas involved in many biological processes of animals, plants and microbes. Previous work has demonstrated that NO is formed during hypoxia in alfalfa ( Medicago sativa L.) root cultures and that the levels of NO detected are inversely related to the levels of expression of class-1 hemoglobin expressed in the tissue. The objectives of this study were: to examine whether NO is produced in transgenic maize ( Zea mays L.) cell-suspension cultures exposed to anoxic growth conditions; to determine whether a similar relationship existed between a class-1 hemoglobin and the amount of NO detected under these conditions; and, to estimate the route of formation and breakdown of NO in the tissue. Maize cell-suspension cultures, transformed to express the sense or antisense strands of barley hemoglobin were used to overexpress or underexpress class-1 hemoglobin. A maize cell-suspension culture transformed with an empty vector was used as a control. Up to 500 nmol NO (g FW)(-1) was detected in maize cells exposed to low oxygen tensions for 24 h. The steady-state levels of NO in the different cell lines under anoxic conditions had an inverse relationship to the level of hemoglobin in the cells. There was no detectable NO produced under aerobic growth conditions. Spectroscopic data demonstrated that recombinant maize hemoglobin reacted with NO to form methemoglobin and NO(3)(-). Nitrate was shown to be a precursor of NO in anoxic maize cell-suspension cultures by using (15)NO(3)(-) and electron paramagnetic resonance spectroscopy, suggesting that NO is formed via nitrate reductase during hypoxia. The results demonstrate that NO is produced in plant tissues grown under low oxygen tensions and suggest that class-1 hemoglobins have a significant function in regulating NO levels.