The detection of antidrug specific IgE in serum is usually performed by a sandwich-type immunoassay in which the serum IgE is first adsorbed to a reactive phase and subsequently quantified via the binding of an anti-IgE tracer. The preparation of a new drug-reactive phase requires one to establish carefully different steps of validation: 1) criteria of positivity of control sera 2) competitive inhibition assays with the soluble drug, which should include the determination of the inhibition constant rather than estimation of a single inhibition percentage, especially when the assay is performed for the identification of determinants 3) estimation of nonspecific binding of IgE to the solid phase, including hydrophobic binding. The competitive inhibition depends on the concentration of the competitor and of IgE in the test-tube and the concentration of reactive drug bound to the solid phase. We have improved the inhibition assay by performing the Dixon test for calculating the inhibition constant (Ki) of the competitor. The Ki of six different muscle relaxants was determined in 12 patients who experienced an anaphylactic reaction to muscle relaxants. The values ranged between 1.5 nM and 2.5 microM. This confirmed the great heterogeneity of drug IgE cross-reactivity among patients. The Ki value of the incriminated drug was the lowest (affinity, the highest) in eight of the 12 patients. It was better correlated to clinical data than the classical inhibition assay. A hydrophobic environment seemed to be necessary, close to the quaternary ion, to allow IgE binding to the muscle relaxant. By contrast, in tiemonium, a hydroxyl group present at a distance of about 3 A from the quaternary ion may explain why this molecule had a high Ki (microM). In conclusion, it should be recommended, in molecular-recognition studies, that the inhibition constant of the soluble drug and of the related compounds be determined to complement the experiments based only on hapten inhibition assays.