Replicative senescence limits the proliferation of somatic cells passaged in culture and may reflect cellular aging in vivo. The most widely used biomarker for senescent and aging cells is senescence-associated β-galactosidase (SA-β-gal), which is defined as β-galactosidase activity detectable at pH 6.0 in senescent cells, but the origin of SA-β-gal and its cellular roles in senescence are not known. We demonstrate here that SA-β-gal activity is expressed from GLB1, the gene encoding lysosomal β-D-galactosidase, the activity of which is typically measured at acidic pH 4.5. Fibroblasts from patients with autosomal recessive GM1-gangliosidosis, which have defective lysosomal β-galactosidase, did not express SA-β-gal at late passages even though they underwent replicative senescence. In addition, late passage normal fibroblasts expressing small-hairpin interfering RNA that depleted GLB1 mRNA underwent senescence but failed to express SA-β-gal. GLB1 mRNA depletion also prevented expression of SA-β-gal activity in HeLa cervical carcinoma cells induced to enter a senescent state by repression of their endogenous human papillomavirus E7 oncogene. SA-β-gal induction during senescence was due at least in part to increased expression of the lysosomal β-galactosidase protein. These results also indicate that SA-β-gal is not required for senescence.