Stepwise selection for increased mefloquine resistance in a line of Plasmodium falciparum in vitro resulted in increased resistance to halofantrine and quinine, increased sensitivity to chloroquine, and amplification and overexpression of the P-glycoprotein gene homolog (pfmdr1). A point mutation (tyrosine to phenylalanine) noted at amino acid 86 in pfmdr1 in the mefloquine-resistant line W2mef was amplified in more resistant lines derived from it by in vitro selection pressure with mefloquine. Conversely, lines selected for increased chloroquine resistance exhibited a revertant phenotype that was sensitive to mefloquine and halofantrine. These lines also demonstrated increased sensitivity to quinine, loss of amplification of pfmdr1, loss of the mefloquine/halofantrine phenylalanine-86 mutation, and selection for a tyrosine-86 mutation previously associated with chloroquine resistance. These findings provide strong evidence for pfmdr1 mediating cross-resistance to halofantrine and mefloquine in P. falciparum in vitro.