Oxidized and cross-linked proteinacious materials (lipofuscin, age pigments, ceroid, etc.) have long been known to accumulate in aging and in age-related diseases, and some studies have suggested that age-dependent inhibition of the proteasome and/or lysosomal proteases may contribute to this phenomenon. Cell culture studies trying to model these aging effects have almost all been performed with proliferating (divisionally competent) cell lines. There is little information on nondividing (postmitotic) cells; yet age-related accumulation of oxidized and cross-linked protein aggregates is most marked in postmitotic tissues such as brain, heart, and skeletal muscles. The present investigation was undertaken to test whether oxidized and cross-linked proteins generally accumulate in nondividing, IMR-90 and MRC-5, human cell lines, and whether such accumulation is associated with diminished proteolytic capacities. Since both protein oxidation and declining proteolytic activities might play major roles in the age-associated accumulation of intracellular oxidized materials, we tested for protein carbonyl formation, proteasomal activities, and lysosomal cathepsin activities. For these studies, confluent, postmitotic IMR-90 and MRC-5 fibroblasts (at various population doubling levels) were cultured under hyperoxic conditions to facilitate age-related oxidative senescence. Our results reveal marked decreases in the activity of both the proteasomal system and the lysosomal proteases during senescence of nondividing fibroblasts, but the peptidyl-glutamyl-hydrolyzing activity of the proteasome was particularly inhibited. This decline in proteolytic capacity was accompanied by an increased accumulation of oxidized proteins.