Nerve growth factor (NGF) can regulate nitric oxide synthase (NOS) expression and nitric oxide (NO) can modulate NGF-mediated neurotrophic responses. In this study, the role of NO in NGF-stimulated amyloid precursor protein (APP) levels was studied. PC12 cells were treated with either the non-selective NOS inhibitor N(omega)-nitro-L-arginine methylester (L-NAME) or the inducible NOS selective inhibitor s-methylisothiourea (S-MIU), and the effect on NGF-mediated increases in APP expression was determined. NGF significantly increased total APP protein levels following 96 h of treatment and this increase was prevented in cells pre-treated with S-MIU. Pre-treatment of cells with actinomycin D also blocked this NGF-mediated induction of APP, indicating de novo protein synthesis is necessary. Treatment with NGF increased APP promoter activity; however, this increase was only partially inhibited by pre-treatment with S-MIU and was increased in the presence of L-NAME. This suggests that NO may be modulating other aspects of APP expression in addition to transcription. Inhibition of NGF signaling pathways was also investigated using inhibitors of mitogen-activated protein (MAP) kinase (U0126), Akt (LY294002) and protein kinase C (PKC; U73122 and bisindolylmaleimide 1 (BIS-1)) activation. Inhibition of each of these pathways prevented NGF-mediated increases in APP protein expression; however, only BIS-1 attenuated NGF-mediated increases in promoter activation. This study indicates that NO is involved in the NGF-mediated regulation of APP, in part at the level of APP transcription and could involve the modulation of NGF signal transduction pathways.