The cellular response to ionizing radiation (IR) includes induction of the c-jun and EGR1 early response genes. The present work has examined potential cytoplasmic signaling cascades that transduce IR-induced signals to the nucleus. The results demonstrate activation of the 40S ribosomal protein S6 kinase, pp90rsk, in human U-937 myeloid leukemia cells. Partial purification of pp90rsk by affinity chromatography demonstrated an increase in S6 peptide phosphorylation when comparing irradiated with control cells. IR-induced activation of pp90rsk was further confirmed in immune-complex kinase assays. In contrast to these findings, there was no detectable induction of pp70S6K. Previous work has demonstrated that mitogen-activated protein kinase activates pp90rsk. The present results further show that IR treatment is associated with induction of mitogen-activated protein kinase activity and that this event is temporally related to activation of pp90rsk and early response gene expression. These findings suggest that activation of the mitogen-activated protein kinase/pp90rsk cascade is involved in the response of cells to IR exposure.