The aim of this study was the identification of the essential structural elements in the 12-formyl-5,6-dihydroindolo[2, 1-a]isoquinoline system required for the inhibition of tubulin polymerization which is understood to be the predominant mode of action of this class of cytostatics. Since 2-phenylindole forms the main fragment of this tetracycle, it was used as the basic structure and modified with respect to the number and positions of the oxygen functions in the aromatic rings. Further modifications related to the nitrogen, which was both replaced by oxygen and sulfur and alkylated. All derivatives were tested for cytostatic activity in human breast cancer cells (MDA-MB 231, MCF-7) and inhibition of tubulin polymerization. The spectrum of activity ranged from inactive to IC50 values of 35 nM (cell growth inhibition) and 1.5 microM (tubulin polymerization), respectively, for the most active derivative 3e (3-formyl-6-methoxy-2-(4-methoxyphenyl)indole). Although the correlation between antiproliferative activity and inhibition of tubulin polymerization was not very pronounced, all of the potent cytostatic agents in this study disrupted microtubule assembly completely at the standard concentration of 40 microM. By fluorescence microscopy it was demonstrated that the derivative 3e degrades the cytoskeleton in a similar fashion as colchicine does leading to the condensation of the microtubules around the nucleus after treatment. The comparison between hydroxy and methoxy derivatives revealed a striking difference between the 2-phenylindole derivatives and the indoloisoquinolines. In the 2-phenylindole series, the methoxy compounds were much more effective than the free phenols, whereas in the tetracyclic system the effect of the hydroxy derivatives exceeded that of the methylated compounds by 1 order of magnitude. Preliminary studies on the binding mode showed that both the 2-phenylindole derivatives and the indoloisoquinolines bind to the colchicine site on tubulin.