As part of our continuing search for potential anticancer drug candidates in the 2-aryl-1,8-naphthyridin-4-one series, we have synthesized a series of substituted 2-thienyl-1, 8-naphthyridin-4-ones. Most compounds showed significant cytotoxic effects (log GI(50) < -4.0; log molar drug concentration required to cause 50% growth inhibition) against a variety of human tumor cell lines in the National Cancer Institute's in vitro screen, including cells derived from solid tumors such as non-small-cell lung, colon, central nervous system, melanoma, ovarian, prostate, and breast cancers. The most active compounds (31-33,40) demonstrated strong cytotoxic effects with ED(50) values in the micromolar or submicromolar range in most of the tumor cell lines. The most cytotoxic compounds inhibited tubulin polymerization at concentrations substoichiometric to the tubulin concentration. The most potent inhibitors of polymerization (40,42,43) had effects comparable to those of the potent antimitotic natural products podophyllotoxin and combretastatin A-4 and to that of NSC 664171, a particularly potent, structurally related analogue. Only compound 40 was a potent inhibitor of the binding of radiolabeled colchicine to tubulin, and it was both the most cytotoxic agent and the most effective inhibitor of polymerization among the newly synthesized compounds.