The fine specificity of anti-hapten antibodies was quantitatively characterized in terms of the capacity of a series of nitrophenyl ligands to inhibit the binding of 3H-2,4-dinitrophenyl-∈-aminocaproic acid (3H-DNP-EACA) by anti-DNP antibody. Applying this assay to antisera produced by mice, rats, guinea pigs and rabbits in response to immunization with DNP keyhole limpet hemocyanin (DNP-KLH), species-specific binding characteristics were noted. These differences involve the relative affinity and relative slopes of inhibition for dinitrophenol, trinitrophenyl ∈-aminocaproic acid and dinitroaniline. Anti-DNP antibodies produced by rabbits and guinea pigs in response to immunization with DNP-bovine ∈ globulin resemble those produced by these species in response to DNP-KLH immunization. Thus, different mammalian species produce antibody populations with different patterns of cross-reactivity in response to the same antigenic stimulus.