Experiments were performed to determine the role of acetylcholine (ACh) in neuromuscular transmission in the heart of the leech Hirudo medicinalis. Superfused or iontophoretically applied ACh rapidly depolarized both isolated heart muscle cells and muscle cells in isolated hearts in a dose-dependent manner. the depolarization was associated with a conductance increase of the muscle membrane that had a reversal potential of -9 mV. Eserine potentiated the response to superfused ACh, reducing the threshold from 10(-6) to 10(-8) mol l-1. Acetylcholinesterase was localized histochemically to be in the immediate area of neuromuscular terminals. Superfused nicotinic agonists mimicked the effects of ACh, while superfused nicotinic antagonists reversibly blocked the iontophoretic response of heart muscle fibres to ACh. 5 X 10(-7) mol l-1 curare, 5 X 10(-5) mol l-1 nicotine and 1 X 10(-4) mol l-1 atropine reduced the iontophoretic response to half its original amplitude. Alpha-bungarotoxin did not block the response of heart muscle cells to iontophoretically applied ACh. Curare was used to determine whether the neurones that innervate the heart-HE motor neurones and HA modulatory neurone--use ACh as a neuromuscular transmitter. The fast depolarizing component of the HE cell's neuromuscular transmission was reversibly blocked by 10(-4) mol l-1 curare, while the HA cell's modulatory effects on the heart were apparently unaffected by 10(-4) mol l-1 curare. Our results indicate that heart muscle cells have nicotinic acetylcholine receptors that open in the presence of ACh, thereby increasing membrane conductance. The HE motor neurone is probably cholinergic and engages these receptors in its neuromuscular transmission, while the HA modulatory neurone is probably not cholinergic.