Cathepsin B (EC 3.4.22.1) and other cysteine proteases are synthesized as zymogens, which are processed to their mature forms autocatalytically or by other proteases. Autocatalytic processing was suggested to be a bimolecular process, whereas initiation of the processing has not yet been clarified. Procathepsin B was shown by zymography to hydrolyze the synthetic substrate 7‐N‐benzyloxycarbonyl‐l‐arginyl‐l‐arginylamide‐4‐methylcoumarin (Z‐Arg‐Arg‐NH‐MEC), suggesting that procathepsin B is catalytically active. The activity‐based probe DCG‐04, which is an E‐64‐type inhibitor, was found to label both mature cathepsin B and its zymogen, confirming the zymography data. Mutation analyses in the linker region between the propeptide and the mature part revealed that autocatalytic processing of procathepsin B is largely unaffected by mutations in this region, including mutations to prolines. On the basis of these results, a model for autocatalytic activation of cysteine cathepsins is proposed, involving propeptide dissociation from the active‐site cleft as the first step during zymogen activation. This unimolecular conformational change is followed by a bimolecular proteolytic removal of the propeptide, which can be accomplished in one or more steps. Such activation, which can be also facilitated by glycosaminoglycans or by binding to negatively charged surfaces, may have important physiological consequences because cathepsin zymogens were often found secreted in various pathological states.