Podophyllotoxin and epipodophyllotoxin react with tubulin at the same binding site as colchicine, but in contrast to colchicine, do not appear to exert their cytotoxicities by mechanisms dependent on P-glycoprotein (Pgp) expression. To investigate structural requirements for Pgp recognition a series of podophyllotoxin and epipodophyllotoxin derivatives have been synthesized. Their interactions with the multidrug resistance-related protein Pgp have been studied by evaluating their relative cytotoxicities versus P388-sensitive murine leukemic cells and a classic multidrug-resistant (MDR) Pgp-overexpressing subline (P388/ADR), and their relative tubulin polymerization inhibitory activities against microtubular proteins have been determined. Based on tridimensional structure-activity relationships within this series of compounds, structural requirements for Pgp recognition have been identified. Moreover, proposals are made for extending these criteria to other chemical classes of anticancer drugs.