The effect of prefeeding Dermacentor andersoni Stiles on naïve and previously challenged cattle before conducting virulence assays by using hamsters was examined. In an initial observational trial, ticks prefed on a previously challenged heifer did not cause paralysis after transfer to hamsters, whereas ticks from the same lot that were prefed on a naïve heifer did cause paralysis. Subsequent paired trails were conducted to simultaneously compare paralysis after feeding on naïve and previously challenged heifers. In the first trial, prefeeding ticks on a previously challenged heifer did not result in loss of virulence. In a second trial, ticks fed on a previously challenged heifer had greatly diminished virulence after transfer to hamsters. This also occurred in a third trial after the previously challenged animal had two prior exposures to virulent D. andersoni. Weight gains of ticks that did not cause paralysis after transfer to hamsters were higher after feeding on previously challenged cattle, indicating that loss of virulence was not due to failure of ticks to reattach and feed. Ticks gained little weight on paralyzed hamsters due to the reduced time spent feeding on hamsters when paralysis occurred. Results indicate that assaying ticks for virulence requires prefeeding on naïve hosts. If these are not available, rabbits, a resistant host, can be used for prefeeding ticks.