After somatic cell fusion between splenocytes of immunized BALB/c mice and NS-1 myeloma cells, eight clones were obtained secreting anti-alprenolol antibodies as characterized by means of an ELISA. Four of these were subcloned and were studied further. The association constant for alprenolol ranged from 1.9 X 10(6) M-1 to 24 to 10(6) M-1. Competitive inhibition of [3H]-l-dihydroalprenolol binding revealed cross-reactivity with beta-adrenergic ligands, with a higher avidity for antagonists than for agonists. Two of the antibodies had a higher affinity for the l-isomer than for the d-isomer. The most stereospecific of these antibodies showed only affinity for beta-adrenergic antagonists and for the agonist isoproterenol. The other recognized both beta-adrenergic antagonists and agonists; it also showed an increase in tryptophan fluorescence after ligand binding. This property was used for the physicochemical study of the hapten-antibody interaction.