Since currently used assays of amphotericin B (AMB) lack sensitivity or are not easily adaptable in all laboratories, we have developed an enzyme immunoassay for AMB in biological fluids and tissues. Antibodies to AMB were raised in rabbits after administration of an AMB-bovine serum albumin conjugate. The enzymatic tracer was obtained by coupling AMB via its amino group to acetylcholinesterase (EC 3.1.1.7). These reagents were used for the development of a competitive immunoassay performed on microtitration plates. The limit of quantification was 100 pg/ml in plasma and 1 ng/g in tissues. The plasma assay was performed directly without extraction on a minimal volume of 0.1 ml. The intra-and interassay coefficients of variation were in the range of 5 to 17%, and the recoveries were 92 to 111% for AMB added to human plasma. The assay was applied to a pharmacokinetic study with mice given AMB intraperitoneally at the dose of 1 mg/kg. The drug distribution in selected compartments (plasma, liver, spleen, lung, and brain) was monitored until 72 h after administration. In conclusion, our assay is at least 100-fold more sensitive than previously described bioassays or chromatographic determinations of AMB and may be useful in studying the tissue pharmacokinetics of new AMB formulations and in drug monitoring in clinical situations.Amphotericin B (AMB) has been considered for more than 30 years the major antifungal drug for serious systemic fungal infections, which, owing to AIDS and improved organ transplant immunosuppression drugs, are becoming tragically frequent in immunocompromised individuals (12,20). AMB's spectrum of activity is broad, since antiviral or antiprion effects have also been reported (12). The clinical value of AMB has been reinforced by incorporation in lipid-based carriers, thus reducing its nephro-and hematotoxicity, and these new formulations have been investigated in animals and humans (4,15,25). In parallel with these recent developments, a need arose for suitable analytical methods to monitor AMB levels in tissue or plasma and thus establish pharmacokinetic and pharmacodynamic relationships. Assays based on in vitro biological activity or chromatographic separations have been described and used to monitor AMB in the circulation (3,11,19,22,23). These methods, however, are characterized by a relatively high limit of detection (Ͼ50 ng/ml) and by technological limitations. These disadvantages may be potentially circumvented by antibody-based technology, and enzyme-linked immunosorbent assays for AMB have been developed (6, 18). Although such techniques allow direct assay in a small sample volume, they have not been applied to tissues and have proved to be poorly sensitive since their limits of quantification are above 150 ng/ ml. In this report we describe the development and application of a competitive enzyme immunoassay which allows measurement of AMB in tissues and plasma with sensitivities 1,000-fold greater than those reported elsewhere. The assay is performed with specific AMB antibodies ...