Using the immuno-fluorescence and immuno-gold electron microscope technology, localization of nitric oxide synthase (NOS)-like proteins was determined in guard cells of Vicia faba L. NOS is mainly localized in nucleus, cytoplasm, chloroplast, mitochondria and the cell wall of guard cells. Scorch and exogenous JA can enhance the level of nitric oxide (NO) and increase NOS activity in both leaf and epidermis, and the changing pattern of NOS activity was consistent with the change of NO. NOS inhibitor, L-NAME, inhibited JA-induced NO generation. From the results, we presumed that NO generation from NOS pathway is the main pathway in the stress and JA responses. The pharmacological experiment showed that increasing the Ca2+ at a suitable concentration promoted leaf NOS activity and the NO level, indicating that NOS activity together with the distribution of NO is Ca2+-dependent. NOS and NO are possibly involved in the regulation of stomatal movement thus playing an important role in plant stress responses.