Tacrolimus is a relatively new immunosuppressant used in organ transplantation to prevent graft rejection. However, its use is not devoid of side effects, making it important to maintain blood concentrations within therapeutic ranges. Several analytical methods are currently available for routine drug monitoring. However, these methods are based on use of the same monoclonal antibody, which also cross-reacts with some metabolites, resulting in overestimation of some blood concentrations. Even though this antibody appears appropriate for therapeutic drug monitoring, no reference method measures only the parent drug, mainly because of the poor absorptivity of tacrolimus in ultraviolet light. We have developed a method displaying an increased specificity towards the unchanged drug, using conventional equipment available in most clinical laboratories. After chromatographic separation of the blood extract, the tacrolimus fraction is analyzed by an automated microparticle enzyme immunoassay (MEIA) performed on the IMx analyzer (Abbott Labs.). This method is linear from 0 to 40 micrograms/L, yields CVs from 8.5% to 18.2%, and has a detection limit of 5 micrograms/L. Tacrolimus concentrations obtained by HPLC-MEIA in hepatic and renal transplant patients are from 47.5% to 18.8% lower than those obtained by MEIA, according to liver function tests and metabolite accumulation, even though no significant differences were observed between the methods for drug-free blood samples supplemented with known amounts of tacrolimus.