Guinea-pig antisera to bovine insulin and proinsulin were analysed and the association constants and the concentrations of combining sites specific for insulin and for the C-chain were determined. Both antisera contained combining sites with higher (Ak(1) sites) and lower (Ak(2) sites) affinities for the cross-reacting antigens.Antisera produced in response to both insulin and proinsulin had similar concentrations of both the more affine and less affine insulin-specific binding sites. Bovine insulin contaminated with traces of proinsulin did not induce antibodies specific for the C-chain.Antisera to proinsulin contained equal amounts of high affinity binding sites specific for the C-chain and for the insulin part of the molecule. After absorption of the insulin-specific antibodies these sera can be used for the immunoassay of proinsulin.