Neurite outgrowth, which is the most distinct process during neuronal differentiation and regeneration, is an essential aspect of neuronal plasticity. PC12 cells have provided a number of clues about the signaling pathways involved in neurite outgrowth. PC12 cells differentiate into neuron-like cells in response to various stimuli, such as nerve growth factor (NGF) and staurosporine. The ERK signaling pathways play important roles in NGF-induced differentiation of PC12 cells. In contrast to NGF, however, the mechanism for staurosporine-induced neurite outgrowth is still unknown. In the present study, therefore, we examined the mechanisms by which staurosporine triggers neurite outgrowth. Images of living cells were obtained using a CCD camera and morphometric analysis was performed using Image-Pro Plus software. To assess direct modulation of Rac1 activity by staurosporine, the amount of cellular GTP-bound Rac1 was measured using the GST-PAK-CRIB binding assay. NADPH oxidase activity was measured by lucigenin chemiluminescence-based assay and the level of intracellular reactive oxygen species (ROS) was measured in cells loaded with DCF-DA using a confocal microscope. The statistical significance of differences between experimental groups was determined using an unpaired Student’s t-test or one-way ANOVA and Bonferroni’s test as appropriate. The results are expressed as means ± standard error of the mean (S.E.M.). Staurosporine caused rapid neurite outgrowth independently of ERK signaling pathways. In contrast, neurite outgrowth in response to 100 nM staurosporine was accompanied by a 227.9 ± 23.9 % increase in Rac1 activity (p<0.05, n = 3), and the Rac1 inhibitor NSC23766 attenuated the staurosporine-induced neurite outgrowth in a concentration-dependent manner. In addition, suppression of Rac1 activity by expression of the dominant negative mutant Rac1N17 also blocked the staurosporine-induced neurite outgrowth of PC12 cells. Staurosporine caused a 75.4 ± 11.9 % increase in NADPH oxidase activity (p<0.05, n = 5) and increased the ROS production approximately by 150 ± 50 % (p<0.05, n = 5), which was prevented by NSC23766 and diphenyleneiodonium (DPI), an NADPH oxidase inhibitor. Pretreatment of cells with DPI attenuated the staurosporine-induced neurite outgrowth by 66.8 ± 4.2 % (p<0.05, n = 5) and exogenous addition of 100 μM H2O2 accelerated the 10 nM staurosporine-triggered neurite outgrowth by 92.3 ± 9.8 % (p<0.05, n = 7). These results indicate that activation of Rac1/NADPH oxidase, which leads to ROS generation, is required for neurite outgrowth induced by staurosporine in PC12 cells.