Removal of one kidney stimulates synthesis of RNA and protein, with minimal DNA replication, in all nephron segments of the remaining kidney, resulting in cell growth (increase in cell size) with minimal cell proliferation (increase in cell number). In addition to the compensatory renal hypertrophy caused by nephron loss, pathophysiologic renal hypertrophy can occur as a consequence of early uncontrolled diabetes. However, the molecular mechanism underlying renal hypertrophy in these conditions remains unclear. In the present study, we report that deletion of S6 kinase 1 (S6K1) inhibited renal hypertrophy seen following either contralateral nephrectomy or induction of diabetes. In wild type mice, hypertrophic stimuli increased phosphorylation of 40S ribosomal protein S6 (rpS6), a known target of S6K1. Immunoblotting analysis revealed that S6K1^-/- mice exhibited moderately elevated basal levels of rpS6, which did not increase further in response to the hypertrophic stimuli. Northern blotting indicated a moderate up-regulation of S6K2 expression in the kidneys of S6K1^-/- mice. Phosphorylation of the eukaryotic translation initiation factor (eIF) 4E-binding protein 1 (4E-BP1), another downstream target of the mammalian target of rapamycin (mTOR), was stimulated to equivalent levels in S6K1^-/- and S6K1^+/+ littermates during renal hypertrophy, indicating that mTOR was still activated in the S6K1^-/- mice. The highly selective mTOR inhibitor, rapamycin, inhibited increased phosphorylation of rpS6 and blocked 60-70% of the hypertrophy seen in wild type mice but failed to prevent the ~10% hypertrophy seen in S6K1^-/- mice in response to uninephrectomy (UNX), although it did inhibit the basal rpS6 phosphorylation. Thus, the present study provides the first genetic evidence that S6K1 plays a major role in the development of compensatory renal hypertrophy as well as diabetic renal hypertrophy and indicates that UNX- and diabetes-mediated mTOR activation can selectively activate S6K1 without activating S6K2.