Glycopyrrolate (GLY) is a synthetic quaternary ammonium anticholinergic compound which offers a number of advantages over atropine, including less arrhythmogenic influence and lack of significant effect upon the central nervous system, eyes and foetus. Five healthy horses (7.4 ± 2.2 years, 462 ± 31.7 kg) were administered 2.5, 5 and 10 μg/kg doses of GLY iv in a randomised and blinded manner with at least 48 h between treatments. The electrocardiogram and heart rate were recorded on an oscilloscopic and chart recorder while gastrointestinal (GIT) motility was assessed by auscultation of 4 abdominal quadrants, assigning a subjective score from 0 (no motility) to 4 (normal motility). Heart rate, which was observed at baseline, 2, 5, 10, 15, 30 min, and 1, 2, 3, 4, 6, 9, 12, 18 and 24 h post GLY, revealed no change with the 2.5 μg/kg dose, and an increase with 5 μg/kg (59%) and 10 μg/kg (109%) for up to 1 h. The 2.5 and 5 μg/kg doses did not prevent the development of second degree atrioventricular block in some horses for up to 15 min, while the 10 μg/kg dose eliminated any pre-existing block within 5 min. There was a complete loss of GIT motility with the 5 and 10 μg/kg doses and partial loss with the 2.5 μg/kg dose. Subsequent return of motility was apparently dose-dependent with 50% of baseline motility score returning in 2.4, 6.4 and 11.5 h with 2.5, 5 and 10 μg/kg doses, respectively. Two horses receiving a 10 μg/kg dose developed abdominal discomfort. Passage of faeces and appetite were not significantly different with any dose. Glycopyrrolate did not induce any change in the pupillary response to a light source at any stage of observation. The results of this study indicate that 5 μg/kg induces a reasonable increase in heart rate without affecting GIT motility for an excessive length of time in awake, healthy, adult, unsedated horses.