Mammalian cells displaying the multidrug resistance (mdr) phenotype are refractory to the toxic effects of a group of unrelated natural product drugs, many of which are used for cancer chemotherapy. The pattern of cross-resistance can be extremely variable among independently selected cell lines, even though such cells are often exposed to only a single drug. The overexpression of P-glycoprotein (pgp), a 150-180-kDa drug efflux pump, has been shown to confer mdr to otherwise drug-sensitive cells; however, the variable nature of cross-resistance indicates that normal pgps alone are unlikely to account for all of the observed cross-resistance phenotypes. In this report, we examined possible factors contributing to cross-resistance diversity in mammalian cells. We show that drug-resistant Chinese hamster lung cells selected during relatively short periods of drug exposure in vitro (less than 6-8 weeks) routinely overexpressed endogenous pgps and predominantly showed a cross-resistance pattern that was similar to that conferred by the introduction and overexpression of the hamster wild-type pgp1 cDNA alone. Longer drug exposure periods at higher drug concentrations, however, led to the selection of cell lines with altered cross-resistance properties. Like the short term clones, these cell lines all overexpressed endogenous pgp. In one case, the altered phenotype was shown to be caused by the acquisition of point mutations within codons 338 and 339 of the pgp1 gene, leading to two adjacent amino acid substitutions within the encoded pgp. Although the basis for the remaining altered phenotypes remains unknown, these results indicate that additional genetic alterations beyond those responsible for the initial acquisition of mdr emerge in the face of increased selective pressure, thus further modifying or complementing the cross-resistance phenotype initially conferred by wild-type pgp.