The pharmacodynamic (PD) action of tacrolimus (FK) within the T-cell is inhibition of calcineurin phosphatase (CaN). Determination of CaN activity provides us with an important PD marker. Eleven renal transplant patients treated with FK were investigated on day 14 following transplantation and 5 years later. Blood samples drawn before as well as 1, 2, 3, and 4 hours after oral intake of FK were analyzed for CaN activity and blood FK concentrations. Twenty healthy subjects had one blood sample drawn for CaN activity, which was measured as the release of (32)P from a phosphorylated peptide. Radioactivity of (32)P was quantitated by liquid scintillation counting with the results converted to units of CaN utilizing a calibration curve. On day 14, we observed significant inhibition of CaN activity at T:1, 2, and 3 compared with the predose level (P = .002; P = .015; P = .015). Furthermore, all measured CaN activities were significantly different from those observed in healthy nonmedicated subjects. In contrast, at 5 years posttransplant only the CaN activity at T:2 was significantly inhibited compared with the predose level (P = .02). Additionally, all CaN activities at this time were not significantly different from CaN activities in the healthy subjects. We were not able to demonstrate individual CaN activity profiles in the patients. The lack of CaN inhibition at 5 years after transplantation despite relevant drug concentrations, probably reflected the lower drug dose used long after transplantation. This result raises the question of whether CaN inhibition is necessary to hold graft function and whether FK possess CaN-independent mechanisms of action.