We have hypothesized previously that a salt-bridge constraint exists in the alpha(1b)-adrenergic receptor (AR). Docking of the agonist epinephrine can disrupt this constraint via competition of its protonated amine, leading to an agonist-induced activation of second messengers. The amino acids, K331 and D125, which comprise this salt-bridge, should be closely associated with each other in the unbound form of the alpha(1b)-AR. This ionic association should stabilize the negative charge of D125, leading to an increase in its acid strength or a decreased pK(a). If the charged state of D125 is important for agonist binding, then changing the type of amino acid at position 331 should decrease the acid strength of D125, leading to epinephrine affinity changes for the alpha(1b)-AR. To test this hypothesis, site-directed mutagenesis was performed at position 331 of the alpha(1b)-AR. The effect these substitutions had on D125 acid strength was quantitated via epinephrine affinity changes calculated from competition binding experiments performed at different pH values. For all mutations of the alpha(1b)-AR where the positive charge at position 331 was eliminated, there was a significant increase in the pK(a) ( congruent with 0.73) of an acidic amino acid(s). In addition, there was an increase in the binding affinity of epinephrine for these mutants that was associated with a gain in the basal production of inositol triphosphates. These results are consistent with an aspartic acid residue as the counterion for K331 of the salt-bridge constraint, which disrupted, is a part of the receptor activation process. Moreover, changes in the pK(a) of D125 were not dependent on the type of amino acid substituted at position 331. This suggests a mechanism in which K331 is no longer influencing D125 after salt-bridge disruption in the wild-type alpha(1b)-AR, but may move to another stabilized position, analogous to what has been suggested for bacteriorhodopsin. Differences from the wild-type receptor in D125 pK(a) for the K331 mutations were used to estimate the free-energy potential of the constraining salt-bridge. This free energy ( congruent with 1 kcal/mol) is significant, but weak enough to be consistent with an activational mechanism where docking of the receptor agonist has sufficient free energy to cause disruption of the salt-bridge.