Some parameters which might affect RAST results, i.e. incubation length, degree of ligand substitution, and total IgE levels, were examined in a RAST system employing diphenylmethane diisocyanate--human serum albumin (MDI-HSA) conjugates as antigens, both in exposed symptomatic and in non-exposed subjects. The reaction equilibrium was reached after 9 and 18 h. respectively, in the first and the second step of the test. A marked correlation with the degree of ligand substitution was observed, the highest sensitivity being achieved with the lowest (2.9) MDI/HSA molar ratio examined. Higher (up to 50) degrees of substitution resulted in progressively lower levels of radioactivity bound and in a loss of specificity, as confirmed by RAST inhibition experiments. At high levels of ligand substitution, high titers of total serum IgE affected RAST results.