Cytomegalovirus (CMV) was isolated from hemic cells from seven of seven patients with acute CMV mononucleosis and from one of six patients with mononucleosis during convalescence. CMV was isolated from the mononuclear leukocyte fraction, the polymorphonuclear leukocyte fraction, or both cell fractions. Virus was not detected in washed erythrocytes, plasma, or leukocyte lysates. Mononuclear leukocytes from patients with acute CMV mononucleosis displayed an elevated level of incorporation of [3H]thymidine on the day of donation compared with that in cells from convalescent patients or normal donors. Responses to pokeweed mitogen and concanavalin A were significantly lower in patients with CMV mononucleosis than in normal donors. There were no significant differences among the acute, convalescent, and normal donor groups in response to phytohemagglutinin or in the one-way mixed leukocyte reaction. These findings suggest that in patients with CMV mononucleosis virus infects and may persist within peripheral blood leukocytes and that the lymphocytes of these patients are selectively hyporesponsive to certain mitogens.