Comparison of c.d. spectra of Zn-insulin with Zn2+-free insulin demonstrated significant differences. It has been proposed that these differences are due to either changes in the structure of insulin monomers within aggregated insulins or the results of insulin aggregation. The effect of Zn2+ on the immunological activity of insulin indicated that the antigenic determinants of insulin were also altered. The apparent loss of immunological activity of monoiodotyrosylinsulin was demonstrated to be due to the loss of Zn2+ rather than the substitution of iodine. The immunological activity of Zn-insulin and Zn2+-free insulin was compared in both the radioimmune and immune haemolysis-inhibition assays by using an identical population of antibodies and concentrations of inhibitor. Relative to Zn-insulin, Zn2+-free insulin had a markedly attenuated immunological activity in the immune haemolysis-inhibition assay, whereas in the radioimmune assay slightly greater immunological activity was observed with the Zn2+-free insulin. These observations are submitted as evidence that the removal of Zn2+ perturbs the conformation of determinants that react with antibodies operative in the immune haemolysis-inhibition assay (immune haemolysis determinants) and has a minimal effect on the conformation of determinants that react with antibodies operative in the radioimmune assay (radioimmune determinants).