In older humans, infusions of endothelial agonists suggest endothelial dysfunction, due in part to less nitric oxide (NO) and prostaglandin (PG) mediated vasodilation, and a shift toward PG mediated vasoconstriction. Ageing can also be associated with lower exercise blood flow (exercise hyperaemia), but the vascular mechanisms mediating this remain unknown. Notably, in young adults, inhibition of NO or PGs during exercise decreases exercise hyperaemia by ~20% and ~12%, respectively. We tested our first hypothesis that in older humans inhibition of NO would decrease hyperemia, but that inhibition of PGs would increase hyperemia by blocking vasoconstrictor PGs. Fifteen older subjects (65±3 years) performed dynamic forearm exercise for 20 minutes (20 contractions min-1). Forearm blood flow (FBF) was measured beat-to-beat with Doppler ultrasound while saline or drugs were infused sequentially via brachial artery catheter in the exercising forearm. After achieving steady state exercise, L-NAME (25 mg) was infused over 5 minutes to inhibit NO synthase. After 2 more minutes of exercise (saline), ketorolac (6 mg) was infused over 5 minutes to inhibit PGs, followed by 3 more minutes of exercise with saline. Drug order was reversed in 7 subjects. L-NAME reduced steady-state exercise hyperaemia by 12±3% in older subjects (P<0.01), whereas ketorolac had no net effect on blood flow (3±6%, P>0.4). The effects of L-NAME and ketorolac were independent of drug order. By comparing these results with our previous results in young adults, we tested our second hypothesis that in older humans inhibition of NO or PGs would have less impact on exercise hyperemia, due to less vasodilation from these signals. Our results suggest that, compared to young adults, in older humans the relative contribution of NO to exercise hyperaemia is reduced ~45% (22±4% vs 12±3%), but the role of PG in mediating vasodilation is lost in ageing human skeletal muscle. Lower exercise hyperaemia in older humans may be mediated in part by less NO and PG mediated vasodilation during exercise.