Using carboxyfluorescein diacetate succinimidyl ester (CFSE)-tagged cells to measure proliferation in vivo, we found that only memory CD8(+) cells from mice older than 18 months gave measurable levels of proliferation and that the proportion of memory CD8(+) T cells able to proliferate in a nonirradiated recipient increased with age. CD8 cells that had proliferated in vivo contained higher levels of CD28 when compared to CD8 cells that had not divided. Cells with high levels of CD28 were preferentially able to divide in nonirradiated recipients. Using ex vivo intracellular staining analysis, we determined that most of the CD8(+) T cells that were capable of dividing in vivo produced IFN-gamma after isolation from recipient mice or their original host. These studies thus document the presence in aged mice of a population of CD28(hi) CD8(+) cells whose ability to proliferate in vivo without antigenic stimulation and to produce IFN-gamma may be involved in immune regulation.