We previously discovered that sensory nerve action potential amplitudes increased during isometric muscle contraction and that this response could be blocked with tourniquet isolation of the contraction source. The hypothesis for this study was that a circulating factor was responsible for this effect. In this prospective study, baseline and post intravenous injection of serial sural nerve action potential recordings were made in the leg of 8 rabbits. The sequence of the injections was randomized: 1) normal saline placebo, 2) 0.01 mg/kg acetylcholine (ACh) 3) 200 mg/kg Na acetate, 4) 260 mg/kg Na lactate, and 5) 20 mg/kg choline. Results showed there was a 3.8 microV increase in the sural nerve response 6 min after ACh injection compared to baseline at rest (p = .01, power = .9, analysis of variance (ANOVA), repeated measures). There were no significant changes in the amplitudes of the sural nerve after injection of the remaining agents or placebo (p = .33 to .81, ANOVA, repeated measures). In conclusion, circulating ACh is the only agent tested thus far that appears to be responsible for this effect. In addition, the amplitude and temporal curve of this response is similar to that seen after exercise in human subjects. The clinical importance of this study is that ACh plays a role in this newly discovered sensory regulatory mechanism controlled by the motor system.