Both Tacrolimus and Sirolimus bind to the same immunophilin FKBP12, however, their mechanism of action is distinct. Sirolimus inhibits TOR which is an enzyme critical to the immune function. TOR inhibition blocks the signal that mediates T-cell proliferation by preventing cell-cycle progression from G1 to S phase In order to determine the bioactivity of Sirolimus we have developed an assay to determine the level of Sirolimus the lymphocyte of transplant patients. The levels obtained were correlated with lymphocyte count. Whole blood samples from patients on Sirolimus were collected in EDTA tubes. Immediately the lymphocytes from 2 ml of blood were separated using 1.5 ml of Ficoll gradient, by centrifugation for 30 minutes at 2500 RPM. The lymphocytes were washed 3 times with PBS and the pellet was suspended in 150 UL of MERI drug extraction solution (Beirut, Lebanon) which was then added to 300 Ul of IMx The lymphocytes cytoplasmic Sirolimus concentrations were measured using the kits supplied from (Abbott). A corresponding whole blood sample from each patient was used to measure blood levels. To determine the level per lymphocytes the value obtained was divided by the number of lymphocytes and is expressed as Pg/Cell. A pharmacokinetic profile for both blood and lymphocytes was constructed for each patient using data corresponding to T0, T1 and T2. The lymphocytes enumeration for T0, T1 and T2, was performed using the Flow Cytometer FACS Calibur from (Becton Dickinson). The average dose was 2.44±0.68 mg/day with a T0 of 8.01±4.5 WBC of 6791±1254 and a lymphocyte count of 1657±268. There was no correlation between the dose, T0 level and the lymphocyte count, or between the T0 and the dose (r2=0.0095 and 0.1697 respectively). Similarly there was no correlation between T1, T2 and lymphocyte count (r2=0.027 and 0.21) respectively. However there was a strong correlation between the B/Cell and the lymphocyte count (r2=0.819). The higher the concentration of the drug the lower the lymphocyte counts. The assay is sensitive to within 0.45 pg/cell, reproducible with a cv of 6.4 for within assay and 7.5 for intra assay. In order to correlate the lymphocyte levels with the observed clinical and pathological conditions a clinical trial is being performed in two countries.