Tacrolimus (FK506) is a macrolide lactone which is used as an immunosuppressant agent for organ transplantation. Patient monitoring during clinical trials initially involved use of extraction/enzyme immunoassays (ELISA) for plasma and whole blood concentrations of FK506. Therapeutic concentrations of FK506 are approximately 0.4-1.2 ng/ml in plasma and 5-20 ng/ml in whole blood. Blood:plasma ratios are variable among patients and range from 11 to 114. A validation and quality assurance program was established to standardize procedures and assure concordance of ELISA analytic results from clinical sites participating in liver/kidney trials. HPLC/ELISA methods have been used for assessing assay specificity. Comparison of the ELISA method with a HPLC-MS procedure (TexMS) shows good correlation, but indicates metabolite inclusion in the ELISA. This method may be of value in conditions of altered FK-506 metabolism. Commercialization of the immunoassay has been made by Incstar with the ELISA and Abbott Laboratories with an IMx procedure; the two methods are generally comparable. Additional methods offering clinical promise are radioreceptor and pentamer complex ELISA assays. There are diverse methods offering sensitive quantitation but with varying specificity for tacrolimus in patient plasma and whole blood.