The response of the arteriolar network in rat cremaster muscle to continuous intraarterial infusion of nicotine was studied. Measurements were made of mean femoral arterial pressure, inside vessel diameter, red blood cell velocity and volumetric flow rate in each of four series-coupled arteriolar segments. Nicotine was continuously infused in a cumulative fashion in doses of 12.5, 25, 50 and 100 micrograms/kg/min. After a 10 min infusion of each dose, measurements were made again in each arteriole and compared with the values obtained prior to infusion of nicotine. Arterial pressure increased in a graded fashion with increasing dose of nicotine up to 50 micrograms/kg/min. The smaller arterioles demonstrated a dose dependent vasoconstriction and reduction in flow rate which were maximal at a dose of 50 micrograms/kg/min. In another series of experiments, the microvascular responses to nicotine infusion were obtained in the acutely denervated microvasculatured. The nicotine-induced flow reduction was significantly diminished by denervation. In a separate series of experiments nicotine was infused at doses of 25 and 50 micrograms/kg/min, and plasma catecholamine concentrations were determined. Plasma norepinephrine and epinephrine were significantly elevated at only the higher dose. Responses in denervated tissues suggest that plasma catecholamine concentrations were approximately threshold for arteriolar responses. It is concluded that the nicotine-induced flow reduction in rat skeletal muscle is due primarily to enhanced release of norepinephrine from vasomotor nerves with little or no influence from circulating catecholamines.