In the course of synaptic transmission in the brain and periphery, acetylcholine receptors (AChRs) rapidly transduce a chemical signal into an electrical impulse. The speed of transduction is facilitated by rapid ACh association and dissociation, suggesting a binding site relatively non-selective for small cations. Selective transduction has been thought to originate from the ability of ACh, over that of other organic cations, to trigger the subsequent channel-opening step. However, transitions to and from the open state were shown to be similar for agonists with widely different efficacies. By studying mutant AChRs, we show here that the ultimate closed-to-open transition is agonist-independent and preceded by two primed closed states; the first primed state elicits brief openings, whereas the second elicits long-lived openings. Long-lived openings and the associated primed state are detected in the absence and presence of an agonist, and exhibit the same kinetic signatures under both conditions. By covalently locking the agonist-binding sites in the bound conformation, we find that each site initiates a priming step. Thus, a change in binding-site conformation primes the AChR for channel opening in a process that enables selective activation by ACh while maximizing the speed and efficiency of the biological response.