Analytical pharmacology draws heavily on the concept of equilibrium of agonist and silent antagonist concentrations competing at a specific receptor site. This condition breaks down in nerve transmission when transmitter release is inhibited by prejunctional α2-adrenoceptors activated by an agonist such as clonidine. We have developed a method that allows the agonist dissociation constant KA of clonidine to be determined in a robust isolated right atrial assay of mouse, rat and guinea pig. By applying low numbers of field pulses 1–4 to prevent autoinhibitory feedback, clonidine shifted the nerve pulse stimulation-tachycardia curves to the right. These peak responses to field pulses were equated to responses to exogenous noradrenaline and the pKA determined by global fitting and display in the Clark plot. The pKA for clonidine ranged from 8.95 in the mouse, 7.8 in rat and 8.3 in guinea pig. The propranolol pKB was 8.87 in mouse and 8.91 in rat atria, reading very similarly to those values from β-adrenoceptor agonist assays under equilibrium conditions. In mesenteric resistance arteries mounted in a myograph for electrical field stimulation, clonidine again inhibited contractions to field pulses in mouse arteries with a pKA of 7.12, but was inactive in rat arteries due to competing autoinhibitory feedback from nerve-released noradrenaline. In both species, prazosin inhibited the field pulses with a pKB of 9.08 in rat and 9.03 in mouse arteries. We conclude that pKB for antagonists and pKA for the prejunctional inhibitors of nerve transmission can be determined with this novel analytical approach.