Acetylcholine was found here to be a strong modulator of swimming activity in the isolated spinal cord preparation of the adult lamprey (Ichthyomyzon unicuspis). During fictive swimming induced with either D-glutamate or N-methyl-D-aspartate, addition of acetylcholine (200 µM) significantly reduced the cycle period of ventral root bursts to 54%, intersegmental phase lag to 32%, and ventral root burst proportion to 80% of control levels. Effects of acetylcholine were apparent at concentrations as low as 1 µM. Both nicotinic and muscarinic receptors are involved, as application of either nicotinic or muscarinic agonists alone significantly reduced cycle period. There is sufficient endogenous acetylcholine in the spinal cord to modulate ongoing fictive swimming as shown by application of the cholinesterase inhibitor eserine (physostigmine). Eserine (20 µM) significantly reduced cycle period to 78% and phase lag to 58% of control levels, and these effects were reversed with the addition of cholinergic blockers. Addition of only a nicotinic or muscarinic antagonist, mecamylamine (10 µM) or scopolamine (20 µM), respectively, to the spinal cord during fictive swimming produced significant increases in cycle period and phase lag, suggesting that both types of cholinergic receptors participate in endogenous cholinergic modulation. It is concluded that acetylcholine is an endogenous modulator of the locomotor network in the lamprey spinal cord and that acetylcholine may take part in the regulation of cycle period, intersegmental coupling, and ventral root burst duration.