Fifty-eight cell lines in the National Cancer Institute drug screen were analyzed for their ability to efflux the fluorescent dye rhodamine 123 as a functional assay for P-glycoprotein (Pgp). Using flow cytometry, the rhodamine fluorescence was measured for each cell line under four incubation conditions, i.e., after accumulation in the presence or absence of the Pgp antagonist cyclosporin A and after efflux in rhodamine-free medium in the presence or absence of cyclosporin A. The results in some cell lines were compatible with Pgp-mediated efflux. There was a significant correlation between mdr-1 expression and rhodamine efflux in the 58 cell lines (r = 0.788, p = 0.0001). Using the rhodamine efflux data as a seed for COMPARE analysis with the cytotoxicity data on > 30,000 compounds in the National Cancer Institute drug screen database, hundreds of compounds with high correlation coefficients were identified. Selected compounds were tested for reversal of cross-resistance in a multidrug-resistant cell line. A high degree of reversibility, up to 10,000-fold, for some of the compounds was noted in the presence of the Pgp antagonist PSC 833. This finding suggested that compounds with predominately Pgp-mediated resistance were being identified. Using these compounds as seeds for COMPARE analysis against a more restricted database of 187 standard agents, a series of standard compounds were repeatedly identified as having high correlation coefficients with the newly identified Pgp substrates. These standard agents, including phyllanthoside, bisantrene, and homoharringtonine, constitute an mdr-1 profile. New agents identified as being highly correlated with these compounds may benefit from clinical trials with Pgp antagonists.