A series of 2-substituted naphthazarin derivatives, 5,8-dihydroxy-1,4-naphthoquinone (DHNQ) derivatives and 5.8-dimethoxy-1.4- naphthoquinone (DMNQ) derivatives, were synthesized, and their cytotoxic activity against some cancer cell lines and antitumor action against S-I80 tumor were evaluated. In general, 2-( I -hy-droxyalky1)-DHNQ derivatives showed a higher cytotoxicity than 2-( 1 -hydroxyalkyl)-DMNQ derivatives, implying a predominent role of redox cycling rather than electrophilicity in cytotoxicity. 2-( 1 -Alkoxy-4-methylpentyl) or 2-( 1 -acyloxy-4-methylpentyl) derivatives were produced by alkylation or acylation at the C-1' position of 2-( 1 -hydroxy-4-methylpentyl)-DHNQ or DMNQ derivatives. Although the cytotoxicity differed according to the size of the alkyl or acyl chain, alkylation or acylation at the C-1' position did not improve the cytotoxicity remarkably, and DHNQ derivatives were still more cytotoxic than DMNQ derivatives. Separately, in vivo testing showed that 2-( I-acyloxyalky1)-DHNQ derivatives or 2-( I-alkoxyalky1)-DHNQ derivatives expressed a higher antitumor action than 2-( I-hydroxyalky1)-DMNQ or -DHNQ derivatives in contrast to the cytotoxicity observations. The total size of two side chains at C-I' seemed to govern the antitumor activity, with 9 to 11 carbon atoms being optimal. Thus, it is suggested that the physical properties as well as the chemical reactivity are to be considered in relation to the antitumor action of 2-substituted naphthazarin compounds.