Resistance to mefloquine in Plasmodium falciparum has begun to occur along the border of Thailand and Kampuchea. As a means of assessing the natural occurrence of mefloquine resistance, the admission and post-treatment parasite isolates from a mefloquine treatment failure were cloned and characterized. Clones from the admission isolate were susceptible to mefloquine in vitro (ID50 of 3.4 [2–5], G [95% CI] ng/ml) and showed a mixture of isozyme types for glucose phosphate isomerase (GPI types I and II). The post-treatment clones were resistant to mefloquine in vitro (ID50 of 17.3 [13–23] ng/ml) with only one isozyme (GPI type I) detected. These observations suggest that under mefloquine pressure a resistant parasite population was selected in the patient, indicating that the potential for mefloquine resistance already exists in the indigenous P. falciparum gene pool. In addition, the mefloquine-resistant clones showed decreased susceptibility in vitro to halofantrine suggesting possible cross-resistance to this new antimalarial drug currently under development.