Cell-mediated immunity to murine cytomegalovirus (CMV) was tested by an in vitro lymphocyte proliferation assay. CMV antigen was prepared from infected mouse embryo fibroblasts by ultracentrifugation. Control antigen was prepared in a similar fashion from uninfected fibroblasts. Splenic lymphocytes from CMV-infected mice underwent significant proliferation when incubated with CMV antigen. Lymphocytes from uninfected mice did not proliferate when incubated in vitro with CMV antigen. The proliferation was shown to be specific, since lymphocytes from CMV-infected mice did not proliferate when incubated with control antigen or an indifferent (encephalomyocarditis) viral antigen. Lymphocyte proliferation was directly related to the number of lymphocytes in each culture and to the amount of viral antigen. However, at higher numbers of cells and with large amounts of viral antigen, the backgrounds increased, and the standard errors were larger. The optimal incubation time was eight days. Lymphocyte proliferation reached a maximum 15 days after CMV infection but was still significantly elevated above background 60 days later. This type of lymphocyte proliferation test with human lymphocytes and human CMV antigen can be used to study clinical questions regarding cell-mediated immunity to CMV in immunosuppressed patients, in children with congenital CMV, and in transplant patients after vaccination with attenuated CMV.