SUMMARY Uphill running requires more energy than level running at the same speed,largely due to the additional mechanical work of elevating the body weight. We explored the distribution of energy use among the leg muscles of guinea fowl running on the level and uphill using both organismal energy expenditure(oxygen consumption) and muscle blood flow measurements. We tested each bird under four conditions: (1) rest, (2) a moderate-speed level run at 1.5 m s–1, (3) an incline run at 1.5 m s–1 with a 15% gradient and (4) a fast level run at a speed eliciting the same metabolic rate as did running at a 15% gradient at 1.5 m s–1(2.28–2.39 m s–1). The organismal energy expenditure increased by 30% between the moderate-speed level run and both the fast level run and the incline run, and was matched by a proportional increase in total blood flow to the leg muscles. We found that blood flow increased significantly to nearly all the leg muscles between the moderate-speed level run and the incline run. However, the increase in flow was distributed unevenly across the leg muscles, with just three muscles being responsible for over 50% of the total increase in blood flow during uphill running. Three muscles showed significant increases in blood flow with increased incline but not with an increase in speed. Increasing the volume of active muscle may explain why in a previous study a higher maximal rate of oxygen consumption was measured during uphill running. The majority of the increase in energy expenditure between level and incline running was used in stance-phase muscles. Proximal stance-phase extensor muscles with parallel fibers and short tendons, which have been considered particularly well suited for doing positive work on the center of mass, increased their mass-specific energy use during uphill running significantly more than pinnate stance-phase muscles. This finding provides some evidence for a division of labor among muscles used for mechanical work production based on their muscle–tendon architecture. Nevertheless, 33% of the total increase in energy use (40% of the increase in stance-phase energy use) during uphill running was provided by pinnate stance-phase muscles. Swing-phase muscles also increase their energy expenditure during uphill running, although to a lesser extent than that required by running faster on the level. These results suggest that neither muscle–tendon nor musculoskeletal architecture appear to greatly restrict the ability of muscles to do work during locomotor tasks such as uphill running, and that the added energy cost of running uphill is not solely due to lifting the body center of mass.