Oral acetate supplementation enhances glycogen synthesis in some mammals. However, while acetate is a significant energy source for skeletal muscle at rest in horses, its effects on glycogen resynthesis are unknown. We hypothesized that administration of an oral sodium acetate-acetic acid solution with a typical grain and hay meal after glycogen-depleting exercise would result in a rapid appearance of acetate in blood with rapid uptake by skeletal muscle. It was further hypothesized that acetate taken up by muscle would be converted to acetyl CoA (and acetylcarnitine), which would be metabolized to CO2 and water via the tricarboxylic acid cycle, generating ATP within the mitochondria and thereby allowing glucose taken up by muscle to be preferentially incorporated into glycogen. Gluteus medius biopsies and jugular venous blood were sampled from nine exercise-conditioned horses on two separate occasions, at rest and for 24 h following a competition exercise test (CET) designed to simulate the speed and endurance test of a 3 day event. After the CETs, horses were allowed water ad libitum and either 8 l of a hypertonic sodium acetate-acetic acid solution via nasogastric gavage followed by a typical hay-grain meal (acetate treatment) or a hay-grain meal alone (control treatment). The CET significantly decreased muscle glycogen concentration by 21 and 17% in the acetate and control treatments, respectively. Acetate supplementation resulted in a rapid and sustained increase in plasma [acetate]. Skeletal muscle [acetyl CoA] and [acetylcarnitine] were increased at 4 h of recovery in the acetate treatment, suggesting substantial tissue extraction of the supplemented acetate. Acetate supplementation also resulted in an enhanced rate of muscle glycogen resynthesis during the initial 4 h of the recovery period compared with the control treatment; however, by 24 h of recovery there was no difference in glycogen replenishment between trials. It is concluded that oral acetate could be an alternative energy source in the horse.