Infection with Ehrlichia phagocytophila in white-footed mice is transient and followed by a strong immune response. We investigated whether the presence of acquired immunity against E. phagocytophila precludes white-footed mice from further maintenance of this agent in nature. Mice were infected with E. phagocytophila via tick bite and challenged either 12 or 16 weeks later by Ixodes scapularis nymphs infected with the same agent. Xenodiagnostic larvae fed upon each mouse simultaneously with challenging nymphs and 1 week thereafter. Ticks were tested for the agent by PCR, and the prevalence of infection was compared to that in ticks that fed upon nonimmune control mice. Only 30% of immunized mice sustained cofeeding transmission of E. phagocytophila between simultaneously feeding infected and uninfected ticks, compared to 100% of control mice. An average of 6.3% of xenodiagnostic ticks acquired Ehrlichia from previously immunized mice when fed 1 week after the challenge, compared to 82.5% infection in the control group. Although an immune response to a single infection with E. phagocytophila in white-footed mice provided only partial protection against reinfection with the same agent, the majority of mice were rendered reservoir incompetent for at least 12 to 16 weeks. Immunity acquired by mice during I. scapularis nymphal activity in early summer may exclude a large proportion of the mouse population from maintaining E. phagocytophila during the period of larval activity later in the season.