Abstract: Adverse reactions of the domestic horse to environmental stimuli can be problematic in training and management. Hesitation and alarm reactions to visual features of the ground can occur in both ridden work and when handling horses. To assess the effect of one visual feature (colour) on the behaviour of the domestic horse, the reactions of 16 riding horses to 8 different coloured mats were recorded. The effect of stimulus position on these reactions was assessed by presenting them in two different positions, either on the ground (where the horses had to walk over them) or against a wall (where the horses walked past them). Each colour/position combination was presented twice in order to assess the effect of previous experience. An alleyway was constructed to allow the horses to be tested unconstrained and freely walking throughout. The time taken to traverse the alleyway and the observed reaction to the colour was recorded. Significant differences in both measures were found in relation to the position of the colour and whether the subject had previous experience of that colour/position combination. The initial presentation of the colours on the ground produced the highest percentage of adverse reactions. Certain colours encountered for the first time on the ground (yellow, white, black and blue) were found to cause a greater number of adverse reactions than others (green, red, brown and grey) and an increase in time taken to traverse the alleyway. Although a significant difference in relation to colour was found in the behaviour observed during the second presentation on the ground, no difference was found in relation to the time taken to traverse the alleyway. No significant effect of colour was found when the coloured stimuli were presented against the wall. These findings have important implications for situations where the colour of flooring could be controlled in order to minimise adverse behavioural reactions, in particular during initial training.