In order to produce antibodies for radioimmunochemical analysis of secretin in plasma, 16 rabbits were immunized; two different immunization schemes were followed. All rabbits produced detectable antibodies to secretin. The final dilution of the antisera necessary to bind 50% of 5 fmol [125I]secretin varied from 1:5,000 to 1:2,300,000. The avidity of antisera, expressed by the equilibrium constant, ranged from 5 x 10(6) 1 x mol-1 to 3 x 10(11) 1 x mol-1. Six rabbits produced antisera that displayed avidity sufficient for measurement of the physiologic concentrations of secretin in plasma (Keff greater than 10(11) 1 x mol-1). The equation of Sips was used to evaluate the heterogeneity of the antibodies. Seven of the 16 antibodies to secretin had a heterogeneity index of 0.90 or more. Cross-reactivity with structurally related peptide hormones (glucagon, vasoactive intestinal peptide, gastric inhibitory peptide) was found to be negligible for the six antisera examined (those with the highest avidity). Three of the high-avidity antisera were examined for reactivity with secretin fragments. They all bound the C-terminal secretin tricosa and tetradecapeptides almost as well as pure natural secretin, whereas the reactivity with the N-terminal tetradecapeptide was slightly lower.