Individual young adult, middle-aged, and old C57BL/6J male mice were tested for in vitro generated proliferative and cytotoxic responses to H-2 alloantigens under a variety of sensitization conditions. Proliferation in mixed lymphocyte culture (MLC) had decreased by 14 months of age (middle-aged), whether measured by directly assaying cultures in microtitre plates (micro MLC) or by labelling aliquots taken from large culture tubes (macro MLC). Cytotoxicity did not decline until a later age if sensitization was done in large tubes (macro cell-mediated lympholysis, CML). When cytotoxic activity was assayed by measuring lysis after addition of chromated cells to MLCs in microtitre plates (micro CML), differences were revealed between young and middle-aged animals. However, these conditions were suboptimal for generation of cytotoxicity even in young controls and showed even lower responses in the middle-aged group. It was concluded that proliferation showed an earlier, more severe decline than cytotoxicity with age as the proliferative response had declined by middle-age under all sensitization conditions used. With optimal sensitization conditions, senescent mice (26--30 months) showed a four- to ten-fold decrease in cytotoxicity compared with young adult mice.