A rapid colorimetric non-radioactive assay for the determination of bovine mitogen-induced lymphocyte proliferation in cell culture was evaluated using a novel tetrazolium compound (MTS) and an electron coupling reagent (PMS) provided in the CellTiter 96 kit (Promega). The results of the new method were compared with those of the 3H-thymidine incorporation assay using parallels obtained from the same lymphocyte population. The concentrations used in the cell suspension of primary cultured lymphocytes resulted in a significant signal/background ratio when cells were prepared from peripheral blood, spleen or mesenteric lymph nodes. The same concentrations of thymocytes resulted in a weak signal even for the highest concentrations of mitogen. A good correlation was demonstrated between the results of the two methods. The non-radioactive method performed well in assays in bovine mononuclear cells derived from prolactin-treated or -untreated calves, showing a 50% lower responsiveness to mitogenic stimulation in prolactin-deprived animals.