AbstractAcetylcholine has been suggested as a neurotransmitter released in the Aplysia gill by peripheral afferents of central neurons and by peripheral neurons within the gill. The perfused gill, isolated from the abdominal ganglion, was examined. At concentrations greater than 1 μM, acetylcholine elicited a slowly developing tonic contraction of the afferent vein that reversed upon washout. This effect was observed on both quiescent and active preparations. At concentrations less than 1 μM, acetylcholine perfusion resulted in a reduction of gill tone. The excitatory effect of acetylcholine was reduced 80 and 60% by the cholinergic antagonists atropine and hexamethonium, respectively. The acetylcholine‐evoked contraction was potentiated 2.5‐fold when curare was coinfused. Carbachol did not mimic the excitatory effects of acetylcholine. At all concentrations examined (1–100 μM), carbachol infusion reduced baseline tension, the amplitude of spontaneous contractions and contractions evoked by FMRFamide and dopamine. Contractions evoked by perfusion of p‐chlorophenylthiocyclic AMP were greatly reduced when carbachol was added to the perfusate. Further addition of curare reversibly blocked carbachol inhibition of the cyclic AMP%evoked contractions. These finding suggest that excitatory and inhibitory cholinergic receptors are involved in the regulation of gill contractile behavior by acetylcholine.