Urea activates a characteristic subset of signaling pathways in a tissue-specific fashion, including transcription of immediate early genes through activation of the mitogen-activated protein kinase (MAPK), ERK (extracellular signal-regulated kinase), and activation of its transcription factor substrate, Elk-1. The ability of urea to activate the ERK effector and pivotal regulatory kinase, ribosomal S6 kinase (RSK), was investigated in mIMCD3 renal inner medullary collecting duct cells. Urea upregulated RSK activity in a time-dependent fashion in serum-deprived mIMCD3 cells; the effect was maximal at 5 min. Activation by hypertonic NaCl, in contrast, was negligible at 5 min and peaked at 15 min. Both stimuli induced the nuclear translocation of cytosolic RSK, as determined via immunofluorescence. Importantly, activation of RSK by both solutes was MAPK/ERK kinase (MEK) dependent, as determined by the ability of the specific MEK inhibitor, PD-98059, to abrogate the response. Taken together, these data indicate that urea activates the ERK effector, RSK, in cells of the renal medulla in an ERK-dependent fashion, further emphasizing the functional significance of urea signaling through ERK activation in renal medullary cells.