Fear of humans is a source of stress for Holstein dairy cattle and can result when animals are handled aversively. We used aversion learning techniques to determine which handling practices cattle find most aversive. In an aversion race, the cows are repeatedly walked down a race and treatments applied when they reached the end; the time and force required for cows to walk down the race are measured. The animal learns to associate walking down the race with the treatment received; if the treatment is aversive, the animal will take more time and require more force to reach the end of the race than if the treatment is positive. In experiment 1, 54 cows were assigned to four treatments (hit/shout, brushing, control, and food). Treatments of 2-min duration were applied three times a day for 4 days. Cows on the hit/shout treatment took more time and required more force to walk through the race than cows on other treatments, while brushed cows took longer to move through the race than cows given food. In experiment 2, 60 cows were assigned to five treatments (electric prod, shouting, hitting, tail twist, and control). Treatments of 1-min duration were applied three times a day for 3 days. Cows on the shout and electric prod treatments took more time and required more force to walk down the race than cows on the control treatment. In experiment 3, thirty-six 1-1.5-year-old heifers were assigned to three treatments (hand feeding, gentling, or control) applied as in experiment 2. Treatments did not affect the time or force required to walk down the race. The aversion race successfully discriminated between handling treatments that differ greatly in aversiveness but lacked sensitivity to distinguish between treatments that were similar. Although many procedural factors must be considered, aversion learning techniques are an effective method to determine which handling practices cattle find aversive or rewarding.