The in vitro activity of pyronaridine was evaluated against 62 isolates of Plasmodium falciparum from Libreville, Gabon using an isotopic, drug susceptibility microtest and was compared with amodiaquine, chloroquine, quinine, and halofantrine activities. The mean 50% inhibitory concentration (IC50) values of the 62 isolates from Gabon to pyronaridine was 3.0 nM (95% confidence interval [CI] = 2.1-3.9). Pyronaridine was less potent against chloroquine-resistant isolates than chloroquine-susceptible isolates but more potent than chloroquine against chloroquine-resistant parasites. The cut-off value for in vitro reduced susceptibility to pyronaridine was an IC50 > 15 nM. Two isolates (3%) showed an IC50 > 15 nM. A significant positive correlation was found between the activities of pyronaridine and chloroquine (r2 = 0.26, P < 0.001), pyronaridine and quinine (r2 = 0.36, P < 0.001), pyronaridine and amodiaquine (r2 = 0.55, P < 0.001), and pyronaridine and halofantrine (r2 = 0.50, P < 0.001). This correlation suggests in vitro cross-resistance or at least in vitro cross-susceptibility, which is not necessarily predictive of cross-resistance in vivo. The present in vitro findings require comparison with those of clinical studies.