The end result of cellular autophagocytosis and lysosomal digestion is incompletely understood in regard to the roles of the superoxide anion radical (O2-.). Cultured glial cells sequestered endocytotically two probes that were site-specific to the lysosome vacuome and sensitive to free-radical activities. The Sepharose-4B-polyisoluminol probe emitted chemiluminescent light in proportion to externally injected O2(-).. Bioluminescence measurements of beta-glucuronidase activity in intact glial cell lysosomes was achieved with the second site-specific, enzyme-specific Sepharose-4B-(dodecanate)5'-luciferinylglucuronate probe. Considerable activity was found in the lysosomal vacuome. In conclusion, we suggest that the use of site-specific, chemiluminescent probes can be of importance in the study of free radicals.