A Brucella abortus-soluble antigen was investigated, using in vitro assay of lymphocyte immunostimulation, to determine which concentration of this antigen and which period of incubation of the lymphocyte cultures would induce maximum specific lymphocyte immunostimulation as an additional method for further study of B abortus infection in cattle. Soluble antigen was prepared from autoclaved cells of B abortus strain 1119-3. Peripheral blood lymphocytes were obtained from cattle infected with B abortus and from healthy control cattle not infected with B abortus. The lymphocytes were prepared by the Ficoll-Hypaque density gradient technique, suspended in RPMI 1640 medium (1.5 X 10(6)/ml), cultured with several dilutions of soluble antigen, and incubated. Prior to termination of incubation, cultures were labeled with 1 muCi of [3H]thymidine and, after harvesting, assayed for [3H]thymidine incorporation in DNA by a liquid scintillation spectrometer. Maximum specific immunostimulation of lymphocytes from B abortus-infected cattle was induced in this assay system with 6 days' incubation and 22 microgram of protein/ml/1.5 X 10(6) lymphocytes, using protein content to express concentration of soluble antigen in this system.