To investigate the activity of senescence-associated beta-galactosidase (SA-beta-GAL) in the hippocampus of aging rats. Hippocampi of 6-, 18-, and 24-month-old rats were observed by histochemical staining for SA-beta-GAL and cytochemical staining for SA-beta-GAL in cultured hippocampal neurons. The activity of SA-beta-GAL doubled in hippocampal pyramidal cells of the CA3 region in rats between 6 and 18months (14.57±2.74% vs. 31.66±14.12% SA-beta-GAL-positive, respectively), and reached 50.76±14.41% positive at 24months. The activity of SA-beta-GAL also increased as a function of time upon prolonged culture of cultured hippocampal neurons with 95% of cells being SA-beta-GAL-positive at 20days in vitro. Interestingly, no SA-beta-GAL-positive cells were found in neurons of the hippocampal dentate gyrus, a neurogenic region of the brain, at any age examined. SA-beta-GAL can be used as a senescence biomarker in determining senescent neurons in hippocampal pyramidal cells of the CA3 region in advanced aging.