There is substantial evidence that adenosine activates muscle afferent nerve fibers leading to sympathetic stimulation, but the issue remains controversial. To further test this hypothesis, we used local injections of adenosine into the brachial artery while monitoring systemic muscle sympathetic nerve activity (MSNA) with peroneal microneurography. The increase in MSNA induced by 3 mg intrabrachial adenosine (106+/-32%) was abolished if forearm afferent traffic was interrupted by axillary ganglionic blockade (21+/-19%, n=5, P:<0.05). Furthermore, the increase in MSNA induced by intravenous adenosine was 3.7-fold lower and later (onset latency 20.9+/-4.8 seconds versus 8.5+/-1 seconds) than intrabrachial adenosine. Finally, we used forearm exercise (dynamic handgrip at 50% and 15% maximal voluntary contraction, MVC), with or without superimposed ischemia, to modulate interstitial levels of adenosine (estimated with microdialysis) while monitoring MSNA. Fifteen minutes of intense (50% MVC) and moderate (15% MVC) exercise increased adenosine dialysate concentrations from 0.31+/-0.1 to 1.24+/-0.4 micromol/L (528+/-292%) and from 0.1+/-0.02 to 0.419+/-0.16 micromol/L (303+/-99%), respectively (n=7, P:<0.01). MSNA increased 88+/-25% and 38+/-28%, respectively. Five minutes of moderate exercise increased adenosine from 0.095+/-0.02 to 0.25+/-0.12 micromol/L, and from 0.095+/-0.02 to 0.48+/-0.19 micromol/L when ischemia was superimposed on exercise (n=7, P:=0.01). The percent increase in MSNA induced by the various interventions correlated with the percent increase in dialysate adenosine levels (r=0.96). We conclude that adenosine activates muscle afferent nerves, triggering reflex sympathetic activation.