Abstract We previously reported that hydrogen peroxide (H 2O 2) mediates mitogen activation of ribosomal protein S6 kinase 1 (S6K1) which plays an important role in cell proliferation and growth. In this study, we investigated a possible role of H 2O 2 as a molecular linker in Rac1 activation of S6K1. Overexpression of recombinant catalase in NIH-3T3 cells led to the drastic inhibition of H 2O 2 production by PDGF, which was accompanied by a decrease in S6K1 activity. Similarly, PDGF activation of S6K1 was significantly inhibited by transient transfection or stable transfection of the cells with a dominant-negative Rac1 (Rac1N17), while overexpression of constitutively active Rac1 (Rac1V12) in the cells led to an increase in basal activity of S6K1. In addition, stable transfection of Rat2 cells with Rac1N17 dramatically attenuated the H 2O 2 production by PDGF as compared with that in the control cells. In contrast, Rat2 cells stably transfected with Rac1V12 produced high level of H 2O 2 in the absence of PDGF, comparable to that in the control cells stimulated with PDGF. More importantly, elimination of H 2O 2 produced in Rat2 cells overexpressing Rac1V12 inhibited the Rac1V12 activation of S6K1, indicating the possible role of H 2O 2 as a mediator in the activation of S6K1 by Rac1. However, H 2O 2 could be also produced via other pathway, which is independent of Rac1 or PI3K, because in Rat2 cells stably transfected with Rac1N17, H 2O 2 could be produced by arsenite, which has been shown to be a stimulator of H 2O 2 production. Taken together, these results suggest that H 2O 2 plays a pivotal role as a mediator in Rac1 activation of S6K1.