We examined the effects of Triton X-100, digitonin, sodium dodecyl sulfate, taurocholic acid, and cetylpyridinium chloride on hemoglobin-catalyzed and t-butyl hydroperoxide-induced chemiluminescence. The experimental system contained hemoglobin, luminol, t-butyl hydroperoxide, and different concentrations of detergents (5-100 mg/dl) in TRIS-HCl buffer. Control assays were performed by excluding detergents. Chemiluminescence was detected using a liquid scintillation counter in single photon mode. All concentrations chosen for each detergent reduced the maximum chemiluminescence value and retarded the time that maximum chemiluminescence occurred. The most prominent reduction in maximum chemiluminescence was observed with 50 and 100 mg/dl digitonin. The smallest reduction was observed with 5 mg/dl sodium dodecyl sulfate, without retardation of the time that maximum chemiluminescence occurred. Our aim was to use detergents in membrane-containing experimental systems and hence to identify the detergent with the least effect on chemiluminescence. Our results suggest that sodium dodecyl sulfate is the most suitable detergent for chemiluminescence studies in membrane systems.