Two series of 2',3',4',5,6,7-substituted 2-phenyl-1,8-naphthyridin-4-ones and 2-phenylpyrido[1,2-alpha]pyrimidin-4-ones have been synthesized and evaluated as cytotoxic compounds and as inhibitors of tubulin polymerization. Most 2-phenyl-1,8-naphthyridin-4-ones showed potent cytotoxic and antitubulin activities, whereas 2-phenylpyrido[1,2-alpha]pyrimidin-4-ones showed no activity in either assay. In general, a good correlation was found between cytotoxicity and inhibition of tubulin polymerization in the 2-phenyl-1,8-naphthyridin-4-one series. The 2-phenyl-1,8-naphthyridin-4-ones (44-49) with a methoxy group at the 3'-position showed potent cytotoxicity against most tumor cell lines with GI50 values in the low micromolar to nanomolar concentration range in the National Cancer Institute's 60 human tumor cell line in vitro screen. Introduction of substituents (e.g. F, Cl, CH3, and OCH3) at the 4'-position led to compounds with reduced or little activity and substitution at the 2'-position resulted in inactive compounds. The effects of various A-ring substitutions on activity depend on the substitution in ring C. Compounds 44-50 were potent inhibitors of tubulin polymerization, with activity nearly comparable to that of the potent antimitotic natural products colchicine, podophyllotoxin, and combretastatin A-4. Compounds 44-49 also inhibited the binding of radiolabeled colchicine to tubulin, but the inhibition was less potent than that obtained with the natural products. Further investigation is underway to determine if substitution at the 3'-position and multisubstitutions in ring C will result in compounds with increased activity.