The ability of cows (Bos taurus) to perceive depth has never been experimentally investigated. If cows can perceive depth, the milking pit in commercial milking facilities may be fear provoking for dairy cows, as past research has shown that most land-dwelling species possess an instinctive fear of heights. In the current study, 12 dairy heifers (1-year-old cows) were exposed to a milking pit (depth-exposed group) and 13 heifers (control group) were exposed to a standard change in the environment while they moved through a milking facility over a 5-day treatment period. Heifers in the depth-exposed group showed a higher heart rate (p < .05) and stopped more often (p < .05) than did those in the control group; persistence of heart rate but not of behavioral responses on repeated exposures indicated that some habituation to the depth stimulus had occurred. Depth exposure had no effect on cortisol concentrations or on ease of handling. These results indicate that heifers responded differently to a change in depth than they did to a standard change in the environment and provide evidence of both depth perception and acute fear of heights in cows.