The study examined the effect of 8 weeks of exercise training on ATP signaling in human skeletal muscle of 15 young (25 ± 1 years) and 15 older (72 ± 1 years) recreationally active male subjects. Before training, femoral venous plasma [ATP] was higher (P < 0.05) during low-intensity knee-extensor exercise in the older than the young group. During moderate-intensity exercise, phosphodiesterase-5 (PDE5) inhibition (to increase cGMP signaling) led to a greater increase (P < 0.05) in plasma [ATP] in the older group. The vasodilator response to arterial ATP infusion was lower (P < 0.05) in the older group. After training, plasma [ATP] was similar in the two groups during exercise at both workloads and PDE5 inhibition did not change plasma [ATP] in either group. The vasodilator response to ATP infusion was enhanced with exercise training in the older group only. These findings provide novel evidence for altered regulation of plasma [ATP] during moderate-intensity knee-extensor exercise in aging which can be reversed by exercise training. The observed effect of PDE5 inhibition suggests that altered cGMP signaling may be one underlying mechanism. Lastly, exercise training can reverse the age-related reduction in the vasodilator response to intravascular ATP.