Mouse cytomegalovirus (CMV) was rapidly and completely inactivated at 37 C by formalin diluted 1:4,000. Of 18 mice that received two doses of inactivated mouse CMV, 16 survived challenge with 250% lethal doses of mouse CMV; one of 11 mice receiving two doses of a control preparation survived the challenge (P < 0.005). After challenge with live virus, recognizable morbidity occurred in only four of 16 recipients of inactivated mouse CMV, but the virus was recovered from the salivary glands of 15 animals. Mice with reciprocal prechallenge titers of neutralizing antibody of < 4 demonstrated significantly higher morbidity and mortality than mice with higher antibody titers. Administration of inactivated mouse CMV did not depress responsiveness of spleen cells to phytohemagglutinin. Most mice immunized with inactivated virus developed only mild or subclinical infections after challenge with lethal doses of mouse CMV.