Background Inhibition of P70S6 kinase (P70S6K ) phosphorylation in activated T cells is 1 of the major mechanisms by which rapamycin exerts its immunosuppressive action. Study Design Observational cohort study. Settings & Participants 2 different groups of kidney transplant recipients at a single center: 30 transplant recipients converted from mycophenolic acid and low-dose prednisone plus cyclosporine A to mycophenolic acid and low-dose prednisone plus rapamycin therapy for chronic allograft nephropathy (group 1) and 16 recipients of suboptimal organs converted from tacrolimus plus rapamycin to rapamycin therapy alone after 3 months (group 2). Predictor Exposure to rapamycin therapy and rapamycin trough levels. Outcomes & Measurements Basal and stimulated phosphorylation of P70S6K was measured by using Western blotting in patients’ peripheral-blood mononuclear cells before and 6 to 11 months after conversion to rapamycin-based therapy. Kinase activation was attained in vivo by means of intravenous insulin injection. Results The potency of rapamycin inhibition of P70S6K phosphorylation varied among patients (RAPA blood concentration required to achieve 50% inhibition of P70S6K activation for mitogen-activated kinase, 3.14 to 12.14 ng/mL) and failed to correlate with drug trough levels. The combination of tacrolimus and rapamycin limited the inhibitory effect of the latter drug on P70S6K activation. Limitations Need for additional studies exploring the relationship between P70S6K activity and kidney graft outcome. Exclusion of patients with diabetes. Conclusions Long-term rapamycin treatment inhibits P70S6K phosphorylation in peripheral-blood mononuclear cells without significant correlation with rapamycin trough levels. By measuring in vivo the biological action of rapamycin, the assay may provide potentially relevant information for the clinical management of rapamycin-treated patients.