The 2"-O-, 3"-O- and 2", 3"-di-O-substituted derivatives (4a-p) of etoposide were prepared by nucleophilic substitution of 4'-O-benzyloxycarbonyletoposide (2) followed by deprotection. Controlled reaction (a limited amount of reagents and low temperature) was required for preparing the mono-O-substituted derivatives.In terms of ED₁₂₅ values, doses which show 125% of T/C against P388 leukemia in mice, both the 2"-O-acetate (4a, ED₁₂₅=0.18 mg/kg) and 3"-O-acetate (4b, 0.23 mg/kg) were nearly as active as etoposide (1, 0.19 mg/kg), while the 2", 3"-di-O-acetate (4c, 1.9 mg/kg) was somewhat less potent. In the replacement with other substituents, antitumor activity of the 2"-O-substituted derivatives was affected much more by the difference of the substituents as compared with that of the corresponding 3"-O-substituted derivatives. In the 2", 3"-di-O-substituted derivatives, the activity was decreased additively on the substituents.