Excitatory motor neurons in the leech are cholinergic. By using a combination of intracellular Lucifer yellow injection and indirect immunofluorescence, we localized FMRFamidelike immunoreactivity to a number of the motor neurons innervating longitudinal and dorsoventral muscle in the leech. All excitatory motor neurons innervating longitudinal muscle (cells 3, 4, 5, 6, 8, L, 106, 107, 108) were labeled with an antiserum to FMRFamide, while the inhibitory motor neurons innervating longitudinal muscle (cells, 1, 2, 7, 9, 102) were not. The excitatory motor neuron innervating medial dorsoventral muscle (cell 117) was labeled, while the excitatory motor neuron innervating lateral dorsoventral muscle (cell 109) was not. The inhibitory motor neuron innervating dorsoventral muscle (cell 101) was also labeled. Nerve terminals along dorsoventral muscle were also labeled with the antiserum. FMRFamide was bath applied to strips of longitudinal muscle while recording tension, and the muscle's response was compared to its response to the previously identified neuromuscular transmitter ACh. Brief applications of FMRFamide caused a contraction approximately one-tenth as large as that caused by an equimolar amount of ACh. The muscle response to FMRFamide was unaffected by curare. During extended exposures, FMRFamide caused a maintained contraction in longitudinal muscle without any apparent desensitization of the FMRFamide receptors and occasionally triggered an irregular myogenic rhythm. This extended exposure to FMRFamide caused a post-exposure potentiation of the longitudinal muscle's response to ACh that shorter applications of FMRFamide did not. Thus FMRFamide may act as a transmitter or modulator in cholinergic motor neurons innervating longitudinal and dorsoventral muscles in the leech.