To investigate the role of the glucoside moiety in the biological activity of VP-16 (etoposide; 4'-demethylepipodophyllotoxin-ethylidene-beta-D-glucoside) and VM-26 (teniposide; 4'-demethyl-epipodophyllotoxin-thenylidene-beta-D-glucoside), a number of acetal and ketal derivatives of 4'-demethylepipodophyllotoxin (DMEP)-beta-D-galactoside were synthesized. The compounds synthesized included acetaldehyde, propionaldehyde, 2-thiophenecarboxaldehyde, phenylacetaldehyde and acetone derivatives. In contrast to the glucose derivatives, where the acetal ring is trans to the pyranose ring, in galactose derivatives it is located in the cis position. The activities of the above compounds have been measured in two different biological assays, based on cross resistance towards mutants exhibiting specific resistance to VP-16/VM-26-like drugs and DNA-strand breaks as measured by the alkaline elution technique. All of the above compounds showed specific cross resistance to VpmR mutants (mutants resistant to VP-16 and VM-26) and caused a dose-dependent enhancement in DNA-strand breakage, providing evidence that they possessed the same kind of biological activity as VP-16 and VM-26. The relative activities of the DMEP-galactose derivatives have been compared with the corresponding DMEP-glucoside compounds. These studies reveal that, for the acetal and ketal derivatives with small R groups (acetaldehyde and acetone derivatives), the activities in the two series are comparable. However, for derivatives with larger, more hydrophobic R groups (2-thiophene or phenylacetaldehyde), the glucoside derivatives showed about 8-10-fold higher activity in comparison with the corresponding galactoside compounds.