Tacrolimus is a calcineurin inhibitor (CNI), and the main immunosuppressant used in solid organ transplantation. However, it causes complications such as hypertension, acidosis, hyperkalaemia, diabetes mellitus and hypercalciuria, mirroring the metabolic syndrome, which may be mediated by altered renal tubular transport mechanisms (Jain et al., 1999; Kim et al., 2004). As calcineurin is a protein phosphatase, and tacrolimus has been shown to alter the activity of serine/threonine kinases, we used quantitative phosphoproteomics to identify novel phosphoproteins involved in pathways that lead to the adverse effects of tacrolimus. Male C57BL/6J mice of 6-8 weeks of age (23.4-25.7g) were administrated 2mg/kg/day tacrolimus or vehicle by intraperitoneal injections for two weeks. At the end of the treatment period they were euthanized by cervical dislocation, under Schedule 1, and the kidneys were isolated and removed. The cytoplasmic and membrane fraction of the renal cortices were separated by centrifugation. The proteins were reduced, alkylated and digested by trypsin. Peptides were then labelled with isobaric Tandem Mass Tags, followed by phosphopeptide enrichment. Vehicle and tacrolimus treated samples were mixed together and injected into the liquid chromatography-mass spectrometry (LC-MS/MS) for analysis. LC-MS/MS detected 415 unique phosphopeptides in the cytoplasmic fraction and 622 in the membrane fraction. In both fractions of the renal cortices, ~21% of the phosphopeptides showed a minimum of 10% increase in phosphorylation and ~26.5% showed a minimum of 10% decrease. Several cortical calcium-related proteins were identified in this study and were shown to be highly phosphorylated following tacrolimus treatment. These include adenylyl cyclase 6 (+23.41%), calnexin (+3.54-16.98%), CACNA1E (Voltage-dependent R type Calcium channel) (+22.2%) and Cdhr5 (Cadherin-related family member 5) (+21.64-360.54%). Other distal calcium- transporting proteins, plasma membrane Ca2+ ATPase (PMCA) and the renal sodium-calcium exchanger (NCX1) were quantified using western blot analysis. Values are presented as means ± S.E.M.; statistical significance was calculated using unpaired t-test. Both PMCA and NCX1 showed a significant increase following tacrolimus treatment (NCX1: 1±0.18 vs. 2.76±0.27 tacrolimus treated, n=5, p<0.001), (PMCA: 1±0.14 vs. 3.02±0.24 tacrolimus treated, n=5, p<0.0001). These data suggests that the effects of calcineurin inhibition on renal calcium transport and their potential regulators are complex, profound and may relate to hypercalciuria. Further investigation into the role of these novel candidates in the adverse effects of CNIs is warranted.