A method for the determination of catalase activity (EC 1.11.1.6.) in homogenates and cell suspensions is described by following the decomposition of H2O2 at physiological H2O2 levels. This first chemiluminescence assay for catalase activity is based on the reaction of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) and NaOCl. The chemiluminescence of this reaction specifically depends on the H2O2 concentration and shows fast kinetics of less than 2 s. Using a flow technique, the exponential decay of H2O2 in the presence of catalase is followed down to 10(-8) M H2O2 at pH 7.4 over three orders of magnitude. At these very low H2O2 concentrations neither oxygen is liberated in gaseous form nor enzyme inactivation or loss of cell viability is observed. Addition of the catalase inhibitor NaN3 completely inhibits H2O2 decomposition. Since the method is not influenced by sulfhydryl and amino group containing compounds, it is especially suited for crude tissue homogenates and suspensions of intact cells. Interestingly, application to cell suspensions shows that intact human erythrocytes and rat hepatocytes exhibit only 5.8 and 1.9% of catalase activity when compared to homogenized cells. These data suggest that the diffusion of H2O2 through membranes is lower than that assumed so far.