The tropical horse tick, Dermacentor nitens, is a natural vector of Babesia caballi in the Americas. B. caballi, one of the etiologic agents of equine piroplasmosis, occurs widely throughout the world, but the United States and a few other countries are considered to be free of infection. B. caballi is transovarially transmitted by the one-host tick D. nitens; we tested the hypothesis that B. caballi can persist in multiple generations of D. nitens in the absence of opportunity to reacquire infection from a susceptible equine host. Partially engorged female D. nitens were collected from a B. caballi-infected horse in Puerto Rico and allowed to reattach and feed on an uninfected horse, successfully transmitting the infection. Three subsequent generations of ticks were reared on calves (nonsusceptible hosts for B. caballi), testing for B. caballi infection in each generation by feeding a sample of the larvae on naive horses. The first generation ofD. nitens reared on a nonsusceptible host transmitted B. caballi, whereas the second and third failed to transmit to naive horses, showing that D. nitens infection with B. caballi was restricted to one generation in the absence of alimentary reinfection. These results imply that, in the event of the introduction of this pathogen into areas of the continental United States where D. nitens occurs, the tick could become a short-term reservoir of B. caballi, making control of introduced infections more complex.