The effects of flavonols on P-glycoprotein (Pgp) activity were studied in cultured rat hepatocytes by assessing and transmembrane transport of Rhodamine-123 (R-123) and doxorubicin (DOX). In freshly-plated hepatocytes, containing a low amount of Pgp, flavonols did not affect the cellular retention of DOX, but strongly inhibited the Pgp-mediated efflux of R-123. In 72h-cultured hepatocytes, spontaneously overexpressing functional Pgp, flavonols inhibited R-123 efflux in a dose-dependent manner, but significantly reduced DOX retention while increasing its efflux. A similar effect was found in hepatocytes obtained from rats in which Pgp was induced in vivo by 2-acetamino-fluorene (AAF) or alpha-naphthyl-isothiocyanate (ANIT) treatments. These findings indicate that flavonols, dietary compounds reported to strongly upregulate the apparent activity of Pgp in cancer cell lines, may also modulate differently the transport of putative Pgp substrates in normal rat hepatocytes. The ability to affect the drug-extruding activity at the hepatocyte canalicular membrane could be of relevance to the chemopreventive action of these compounds towards liver carcinogens.