Abstract We have applied a method using the fluorigenic substrate benzoloxycarbonyl-Arg-Arg-amido-4-methylcoumarin to measure cathepsin B, a thiol proteinase, in homogenates of human leukocytes. Data like pH optimum, stability, influence of thiol groups and effects of thiol proteinase inhibitors, lack of binding to Concanavalin A and lack of contribution to the fluorescence by other cathepsins indicate that cathepsin B is the enzyme masured. Although the activity of the enzyme was linear with respect to time at all protein concentrations measured, there was an acceptable 10% deviation of the enzyme activity from linearity as a function of protein concentration. The enzyme in the homogenate was stable at 0 °C but was rapidly inactivated at 50 °C and above pH 6.5–7. Very limited activation on the one hand and variable inhibition on the other was seen by reagents containing thiol groups and thiol proteinase inhibitors respectively. Latency (60% of the enzyme activity) indicates a probable subcellular lysosomal localization. There is no affinity towards the lectin Concanavalin A and the Km value was around 1 mmol/l. Normal enzyme activity values in leukocyte homogenates were determined.