Reflex leg levation habituates during repeated electrical stimulation of mechanosensory afferents in the dactyl of the fifth walking leg of the crayfish, Procambarus clarkii. This was investigated in decerebrate crayfish, and reproduced in an isolated thoracic ganglion preparation. In vivo, trains of stimuli delivered every 2.5 s produced a gradual decrease in the amplitude of the mechanical response, and a concomitant decrease in the number of impulses per burst in the levator muscle myogram. Near complete recovery occurred after 10 min rest, and transient dishabituation was observed after electrical stimulation of the telson. Less frequent or stronger stimuli led to less rapid habituation. In vitro, the same parametric characteristics of habituation were observed in the levator nerve responses, while the intrinsic variability of the reflex was reduced. The response decrement was shown to be unrelated to changes in the afferent excitation. Evoked polysynaptic excitatory postsynaptic potentials (EPSPs) in levator motorneurons decreased in parallel with the levator neurogram. This decrease was unrelated to any change in the resting membrane potential of the levator motorneurons. Intemeurons with habituating EPSPs, antagonistic depressor motorneurons with habituating inhibitory postsynaptic potentials and non-habituating responses in other motorneuronal groups were also found. These findings point to a central locus of habituation upstream from the motorneurons, and offer prospects for a detailed investigation of the mechanisms of habituation in a polysynaptic system.