Cell-mediated immune (CMI) responses in cattle vaccinated with Brucella abortus strain 19 vaccine during calfhood were studied by an in vitro lymphocyte stimulation assay. Cattle were grouped in six groups according to the age after vaccination, and CMI responses of these groups, as well as of individual animals, were compared. Lymphocytes were prepared from peripheral blood of these cattle by the Ficoll-diatrizoate technique. Lymphocytes were then cultured with B abortus-soluble antigen. Culture results were assayed for [3H]thymidine incorporation into DNA by liquid scintillation spectrometry. On a group basis, B abortus-soluble antigen induced lymphocyte stimulation responses in lymphocytes from all the groups, except the sixth group which contained animals that had been vaccinated the longest time (18 to 24 months before this experiment). Animals that had been vaccinated for 3 to 6 months had the highest lymphocyte stimulation response. Seroagglutination tests were conducted simultaneously with the lymphocyte stimulation test, but there was no apparent correlation between the concentrations of humoral antibodies and the CMI responses as measured by in vitro specific lymphocyte stimulation. The lymphocyte stimulation test exhibited significantly higher specificity (P less than 0.005) than the serologic tests.