Tacrolimus (FK506) is a macrolide antibiotic with potent immunosuppressive properties. FK506 is 10- to 100-fold more potent than cyclosporin A in preventing organ rejection and in toxicity. Extreme inter- and intrapatient variability and lack of correlation between drug dosage and drug blood levels necessitate consistent and reliable therapeutic drug monitoring. Previous methods for monitoring drug levels have been lengthy and labor intensive and have required organic solvents for sample extraction. We have developed a manual enzyme-linked immunosorbent assay (ProTrac II ELISA) that employs standardized reagents and a nonorganic solvent extraction and yields good assay sensitivity and precision. The calculated mean sensitivity of the assay was 0.18 ng/ml. Interassay precision ranged from 6.3% to 13.1% (coefficient of variation) for FK506 in whole blood (concentration range, 1.0-25.0 ng/ml). Recovery of the drug from spiked EDTA whole blood was 91-98% over the same concentration range. Mean recovery of the drug from clinical samples spiked with kit standards was 99.5%. Dilutions of high-concentration spiked EDTA whole blood samples and high-concentration samples from patients exhibited linearity of observed versus expected values (y = 0.86x - 1.15; r = 0.99). Comparison of ProTrac II with the INCSTAR ProTrac FK506 ELISA by paired Student's t test showed no statistical difference between the methods (p = 0.46). Comparison of ProTrac II ELISA to the IMx microparticle enzyme immunoassay (MEIA) by linear regression resulted in a line with a slope of 1.22 (r = 0.91). Analysis by t test resulted in a p value < 0.001, indicating that the absolute values obtained in these assays are significantly different. The mean (+/-SD) difference in the absolute values was 4.2 +/- 2.6 ng/ml, with the MEIA values consistently higher. These results indicate that ProTrac II is a sensitive, precise ELISA for the determination of tacrolimus in whole blood; it can be performed in < 4 h.