Seven anti-human insulin monoclonal antibodies (mAb) were produced according to an efficient immunization protocol elaborated in our laboratory. Their affinity constants for the binding to the insulin molecule ranged from 5.0 x 10(8) M-1 to 1.0 x 10(10) M-1 when insulin was in solution and from 6.0 x 10(6) M-1 to 2.5 x 10(8) M-1 when insulin was adsorbed onto the microtiter plate. The antigenic sites on the insulin molecule recognized by these mAbs were mapped using two approaches. MAb pairs capable of binding simultaneously to human insulin in solution (using a two-site ELISA) or adsorbed onto a microtiter plate (using a competitive ELISA) were first sought. Three antigenic regions were defined on the surface of adsorbed human insulin and four on soluble insulin. Two distinct antigenic regions common to both the adsorbed and the soluble forms of insulin were defined by our mAbs. In a second approach, the immunological cross-reactivities of these mAbs with species variants of insulin, chemically modified insulin of known structure and a panel of 78 overlapping nonapeptides covering the entire sequence of human proinsulin were assessed. Evidence was obtained that the epitopes recognized by the mAbs included residues conserved during evolution in the insulin molecule. The epitopic specificity of one group of mAbs (group I) was precisely defined. This group recognized a highly conserved region of the insulin molecule including residues 10-17 of the A chain.