Senescence-associated β-galactosidase is widely used as a biomarker of replicative senescence. However, it remains unknown whether this is a distinct enzyme active at pH 6, and differentially expressed in senescence, or a manifestation of an increase in the classic acid lysosomal β-galactosidase. Here we have investigated the origin of senescence-associated-β-galactosidase activity by modifying the intracellular and lysosomal pH of young and senescent human umbilical vein endothelial cells and examining the effect of these manipulations on the levels of activity, using a flow cytometric assay. Lysosomal alkalinisation with chloroquine or bafilomycin A₁, as well as equilibration of the intracellular milieu to pH 6 with nigericin, caused a profound (92-99%) inhibition of the total intracellular β-galactosidase activity. However, independent of pH alterations, senescent cells showed levels of β-galactosidase activity three-to sixfold higher than young cells. This increase in activity occurred in parallel to an increase in β-galactosidase protein levels. Acridine Orange staining revealed an increase in lysosomal content with replicative age, which correlated with the increase in β-galactosidase. These findings demonstrate that senescence-associated β-galactosidase is a manifestation of residual lysosomal activity at a suboptimal pH, which becomes detectable due to the increased lysosomal content in senescent cells.