To enhance the outstanding biological response shown by the corresponding monomers 4 and 5, two classes of bis-acridine-4-carboxamides, 9, with a linker between the 4,4' positions, and 13, with a linker between the 1,1' positions, have been prepared as DNA-binding and potential antitumor agents. The noncovalent DNA-binding properties of these compounds have been examined using gel-electrophoresis and fluorometric techniques. The results indicate that (i). target compounds intercalate DNA; (ii). the bis derivatives with the optimal linker are considerably more DNA-affinic than corresponding monomers; (iii). overall affinity is sensitive to the nature of the linker, of the chromophores, and of the substituents at 7,7'; (iv). often, the bis derivatives show a marked AT-preferential binding. In vitro cytotoxic potency of these derivatives toward the human colon adenocarcinoma cell line (HT29) is described and compared to that of reference drugs. Structure-activity relationships are discussed. Some highly DNA-affinic and potent cytotoxic compounds, 9b,f and 13b,c, have been selected for the National Cancer Institute (NCI) screening on 60 human tumor cell lines and identified as new leads in the antitumor strategies.