The inhibitor CA-074 and its membrane-permeable derivative CA-074Me have been widely employed to study the biological function of cathepsin B. These compounds have been described as selective inactivators of cathepsin B with barely detectable action on other cathepsins. However, in this study it is demonstrated that CA-074 and CA-074Me also inactivate cathepsin L in the presence of the thiol reagents dithiothreitol and glutathione. In addition, it is also found that CA-074Me inactivates cathepsin B in the presence of dithiothreitol, an inhibition reaction previously not described. Furthermore, it is shown that CA-074 and CA-074Me are able to inhibit cathepsin L within living HL-60 cells and bloodstream forms of Trypanosoma brucei brucei. For HL-60 cells it is also shown that the inhibition of cathepsin L by the inhibitors is due to the reducing intracellular environment. These results suggest that CA-074 and CA-074Me are not suitable to discriminate between cathepsin B and L in vivo. Accordingly, previous demonstrations of the involvement of cathepsin B in physiological and pathological processes probably need re-evaluation.