New derivatives of E‐64 (compound CA‐030 and CA‐074) were tested in vitro and in vivo for selective inhibition of cathepsin B. They exhibited 10000–30000 times greater inhibitory effects on purified rat cathepsin B than on cathepsin H and L; their initial K ₁ values for cathepsin B were about 2–5 nM, like that of E‐64‐c, whereas their initial K ₁ values for cathepsins H and L were about 40–200 μM. In in vivo conditions, such us intraperitoneal injection of compound CA‐030 or CA‐074 into rats, compound CA‐074 is an especially potent selective inhibitor of cathepsin B, whereas compound CA‐030 does not show selectivity for cathepsin B, although both compounds CA‐030 and CA‐074 show complete selectivity for cathepsin B in vitro.