SUMMARY Two studies have focused on potential triggers for the trot–gallop transition in the horse. One study concluded that the transition was triggered by metabolic economy. The second study found that it was not metabolic factors but, rather, peak musculoskeletal forces that determine gait transition speeds. In theory, peak musculoskeletal forces should be the same when trotting up an incline as when trotting at the same speed on the level. Assuming this is the case, we hypothesized that if peak forces determine gait transition speeds then horses should switch from a trot to a gallop at the same speed (i.e. the same critical force) regardless of incline. The aim of the present research was to examine the effects of incline on the trot–gallop transition speed in horses and to re-examine the role of metabolism in determining the trot–gallop transition. Horses ( Equus caballus ) were conditioned to run on a high-speed treadmill prior to data collection. Gait changes were recorded for each horse using a standardized testing protocol on the level and when trotting up a 10% incline. Both maximum sustained trotting speeds and minimum sustained galloping speeds, representing the lower and upper limits of the trot–gallop transition, respectively,were significantly slower when trotting up an incline. After completing collection of gait transition data, the horses were trained to extend their gaits beyond the normal transition speeds, and metabolic data were collected. Maximum sustained trotting speeds were not different from the energetically optimal transition speeds, i.e. the speed at which metabolic rates are the same for both gaits.