The study was designed to evaluate how exposure of Plasmodium falciparum to mefloquine modifies the sensitivity of the parasite to four major antimalarial drugs. A recently culture-adapted strain of P. falciparum was subjected to intermittent drug pressure at three different mefloquine concentrations (2.34, 4.68, and 9.37 ng/ml). Growth was monitored by daily evaluation of parasitemia on thin smears. Drug sensitivity tests were done weekly, using a radioisotope microdilution method. Mefloquine was removed from culture media when decreasing parasitemia was observed, and reintroduced when multiplication reoccurred. Parasite survival was inversely proportional to drug concentrations. The parasites tolerated progressively higher concentrations of mefloquine with prolonged exposure to the drug. Throughout this adaptation, the 50% inhibitory concentration for chloroquine and quinine showed no modification, but it increased considerably for mefloquine, exceeding known levels of resistance. Furthermore, a parallel increased resistance to halofantrine was observed, surpassing the normal range of sensitivity. Cross-resistance between mefloquine and halofantrine shown in this study has now been confirmed by epidemiologic in vitro surveys and clone analysis. These findings may have important in vivo consequences and eventually affect the choice of antimalarial therapy.