An increasing number of studies have proven that the kinase suppressor of Ras (KSR1) functions as a scaffolding protein that coordinates the assembly of a multiprotein complex containing mitogen-activated protein kinase and its upstream regulators. However, a few studies have reported that KSR1 can activate c-Raf-1. Therefore, whether KSR1 possesses a kinase activity has been an unresolved issue until now. pCMV-Tag2b-KSR plasmids were transfected into 293T cells. In vitro autophosphorylation was assayed by autoradiography and in vitro kinase was assayed by reversed-phase high performance liquid chromatography. We observed that wild-type KSR1 (WT-KSR) and N-terminal KSR1 (N-KSR) were phosphorylated, but the C-terminal KSR1 (C-KSR) and vector proteins were not. The high performance liquid chromatography profile showed not only the adenosine diphosphate peak but also the uridine triphosphate peak in the WT-KSR and N-KSR groups; both peaks were considerably more significant in these groups than in the others. The WT-KSR and N-KSR groups exhibited transphosphorylation and autophosphorylation activities, while the other groups revealed almost no activity. Here, we demonstrate the nucleoside diphosphate kinase activity of the KSR1 complex and that this activity can be independent of the C-terminus of KSR1. Additionally, we found the autophosphorylation activity of the KSR1 complex to be extremely weak, suggesting that the KSR1 complex possesses an extremely weak kinase activity irrespective of whether it is nucleoside diphosphate kinase activity or serine/threonine protein kinase activity. These data suggest that the kinase activity of the KSR1 complex is derived from its associated proteins.