Isolated peripheral blood lymphocytes (PBL) from cattle immunized or infected with infectious bovine rhinotracheitis (IBR) virus were cultured in vitro with ultraviolet light-inactivated IBR virus, and the degree of lymphocyte blastogenesis was quantitated by measurement of the uptake of [(3)H]thymidine into acid-insoluble material. Lymphocyte blastogenesis only occurred with PBL from immunized or infected animals. The optimal conditions for lymphocyte blastogenesis were defined. Blastogenesis was specific since cells from animals immunized against IBR failed to react with two other herpesvirus antigens tested, herpes simplex and equine rhinopneumonitis viruses. Blastogenesis could be prevented by reacting IBR antigen with IBR-specific antibody before adding to cultures, but incorporating IBR-specific antibody in the culture medium after adding free antigen failed to inhibit blastogenesis. With intranasally infected animals, lymphocyte blastogenesis was detectable after 5 days, reached peak levels between days 7 and 10, and then declined to low levels by day 19. In contrast, levels of neutralizing antibody were barely detectable on day 7 and reached maximal concentrations on day 19. The lymphocyte blastogenesis assay was emphasized as a convenient and useful in vitro correlate of cell-mediated immunity that should help define the role of cell-mediated immunity in immunity to herpesviruses.