Hypoxia-inducible factor-1alpha (HIF-1alpha) protein is degraded under normoxia by its association to von Hippel-Lindau protein (pVHL) and further proteasomal digestion. However, human renal cells HK-2 treated with 15-deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)) accumulate HIF-1alpha in normoxic conditions. Thus, we aimed to investigate the mechanism involved in this accumulation. We found that 15d-PGJ(2) induced an over-accumulation of HIF-1alpha in RCC4 cells, which lack pVHL and in HK-2 cells treated with inhibitors of the pVHL-proteasome pathway. These results indicated that pVHL-proteasome-independent mechanisms are involved, and therefore we aimed to ascertain them. We have identified a new lysosomal-dependent mechanism of HIF-1alpha degradation as a target for 15d-PGJ(2) based on: (1) HIF-1alpha colocalized with the specific lysosomal marker Lamp-2a, (2) 15d-PGJ(2) inhibited the activity of cathepsin B, a lysosomal protease, and (3) inhibition of lysosomal activity did not result in over-accumulation of HIF-1alpha in 15d-PGJ(2)-treated cells. Therefore, expression of HIF-1alpha is also modulated by lysosomal degradation.