The immunological interrelationships of bovine proinsulin, its related fractions and insulin are described. Proinsulin has been iodinated and the labeled material purified and characterized. Proinsulin and its intermediate fractions, which could be converted to desalanyl insulin by trypsin, reacted more strongly than insulin or the “nonconvertible” insulin-like material with antiserum to purified proinsulin. The order of reactivity was reversed in favor of insulin when an insulin antiserum was used. Preincubation of the antiserum to purified proinsulin with large quantities of insulin allowed the preferential measurement of bovine proinsulin, and the assay was unaffected by further addition of insulin. The isolated C-peptide from proinsulin was effective in displacing I-131-proinsulin from this antibody. Human and porcine proinsulin reacted weakly with antisera to bovine proinsulin. The immunological differences between proinsulins are far greater than among the corresponding insulins, and imply that the C-peptide of proinsulin contains major antigenic determinants. It is probable that specific antisera will be needed for the optimal assay of each species of proinsulin.