Blockade of neurotransmitter-gated ion channels alters the kinetics of ion flux across cell membrane and is a promising approach to modulate synaptic transmission.  We seek to develop blockers that are selective for acetylcholine receptors (AChRs) and have blockade kinetics tunable via structural modifications.  A prototype molecule (1a) was found to both stimulate AChR activities and block the open channel.  Single-channel kinetic analysis of 1a and its monovalent homologue suggests a model in which 1a blocks the AChR via at least two distinct modes: blockade through an agonist binding-directed intramolecular mechanism and blockade directly from bulk solution.  In the latter mechanism, the non-blocking trimethylammoium group presumably interacts with the negatively charged vestibule, providing 1a with extra affinity to the channel.  Our results support the hypothesis that AChR blockade can be directed by agonist binding and provide useful insights into the future design of target-selective, kinetically tunable AChR blockers.