Nitric oxide (NO) regulates the expression of multiple genes but in most cases its precise mechanism of action is unclear. We used baby hamster kidney (BHK) cells, which have very low soluble guanylate cyclase and cGMPdependent protein kinase (G-kinase) activity, and CS-54 arterial smooth muscle cells, which express these two enzymes, to study NO regulation of the human fos promoter. The NO-releasing agent Deta-NONOate (ethanamine-2,2 -(hydroxynitrosohydrazone)bis-) had no effect on a chloramphenicol acetyltransferase (CAT) reporter gene under control of the fos promoter in BHK cells transfected with an empty vector or in cells transfected with a G-kinase I␤ expression vector. In BHK cells transfected with expression vectors for guanylate cyclase, Deta-NONOate markedly increased the intracellular cGMP concentration and caused a small (2-fold) increase in CAT activity; the increased CAT activity appeared to be from cGMP activation of cAMP-dependent protein kinase. In BHK cells co-transfected with guanylate cyclase and G-kinase expression vectors, CAT activity was increased 5-fold in the absence of Deta-NONOate and 7-fold in the presence of Deta-NONOate. Stimulation of CAT activity in the absence of Deta-NONOate appeared to be largely from endogenous NO since we found that: (i) BHK cells produced high amounts of NO; (ii) CAT activity was partially inhibited by a NO synthase inhibitor; and (iii) the inhibition by the NO synthase inhibitor was reversed by exogenous NO. In CS-54 cells, we found that NO increased fos promoter activity and that the increase was prevented by a guanylate cyclase inhibitor. In summary, we found that NO activates the fos promoter by a guanylate cyclase-and Gkinase-dependent mechanism.Nitric oxide (NO) 1 is a pluripotential molecule involved in regulating blood pressure, neurotransmission, and immune function (1). One of its major intracellular targets is the heme group of soluble guanylate cyclase with NO markedly stimulating enzymatic activity and thereby increasing the intracellular cGMP concentration (2, 3). Other NO targets are thiolcontaining proteins, iron sulfur proteins and non-heme iron; in addition, NO can react with oxygen to produce peroxynitrite and hydroxyl radical, both of which can have physiological effects (4).NO regulates the expression of multiple genes including c-fos, junB, heme oxygenase, smooth muscle ␣-actin, vascular endothelial growth factor, vascular cell adhesion molecule-1 (VCAM-1), and mitogen-activated kinase phosphatase-1 (5-10). In regulation of VCAM-1, NO appears to act independently of cGMP because it's effect is not mimicked by cGMP analogs, while in regulation of c-fos and junB, NO and cGMP analogs induce similar changes (5, 6, 10). In cases where NO and cGMP analogs function similarly, it is likely that NO works through activation of soluble guanylate cyclase, but this has not been shown definitively. Moreover, it is not clear which of several cGMP target proteins, e.g. cGMP-dependent protein kinases (G-kinase), cGMP-gated ion channels, cGMP-activated...