Abstract: We carried out a questionnaire survey of the caretakers, using 86 riding horses kept in the Equestrian Park, Tokyo (Japan Racing Association). The questionnaire survey used a 5-point scale and a 3-point scale to assess several caretakers' impressions of each horse's temperament, on the basis of the norm and the horse's tendencies in ordinary care and daily training. Factor analysis of the temperament scores obtained with the 5-point scale questionnaire revealed three mutually independent factors that we named “anxiety”, “novelty seeking” and “understanding”. In order to verify the reliability of this questionnaire survey, a balloon reactivity test was conducted using the same horses. Each horse was introduced into an unfamiliar indoor arena (7 mx12.5 mx3 m) in the center of which two balloons slowly revolved. The horses' responses were assessed by recording changes in their behavior and heart rate (HR) during the 5 min experimental period. By comparing the questionnaire survey and the balloon reactivity test, it was found that the horses evaluated as highly anxious by the caretakers tended to show greater HR increases and defecate more often during exposure to the balloon stimuli than did the other horses. Additionally, the horses assessed by caretakers to have problems with ordinary care and/or training showed greater increases of HR and frequency of defecation in the balloon reactivity test, and the horses assessed as having `a long adaptation time to unfamiliar objects' were found to be unwilling to touch the balloons. Thus, the horses' behavior during the balloon reactivity test was highly consistent with their temperament as determined by the questionnaire. These results suggest that the questionnaire survey would be an effective means to assess equine temperamental traits, especially those related to anxiety.