The pharmacokinetics, oral bioavailability, and ex vivo antimalarial activity of mirincamycin isomers in a healthy rhesus monkey model were assessed to support lead optimization of novel nonhemolytic drugs for radical cure and causal prophylaxis of malaria. Fourteen male rhesus monkeys were randomized to four groups, which included cis and trans isomers by the oral and intravenous routes, with vehicle-only controls for each dosing route. Concentration-time data were collected for 7 days and were analyzed by noncompartmental analysis. cis-Mirincamycin had an absolute oral bioavailability of 13.6%, which was slightly higher than that of trans-mirincamycin (11.7%), but this difference was not statistically significant. There was a statistically significant difference between the area under the concentration-time curve from zero to 48 h (AUC(0-48)) of cis-mirincamycin and that of trans-mirincamycin after oral dosing. When cultured in vitro with the W2 clone of Plasmodium falciparum, the 50% inhibitory concentrations for cis-mirincamycin, trans-mirincamycin, and dihydroartemisinin were 11,300, 12,300, and 2.30 nM, respectively. However, when dosed primate plasma was cultured ex vivo against the W2 clone, both isomers had much greater relative potencies than their in vitro activities relative to results for dihydroartemisinin, an increase of approximately 100-fold for the cis isomer and 150-fold for the trans isomer. Further, oral ex vivo activity was significantly higher than intravenous activity for both isomers, particularly during the first 90 min following dosing, suggesting the first-pass formation of one or more metabolites with blood-stage antimalarial activity. Identification of the metabolic pathways and metabolites may help to further delineate the properties of this class of drugs with previously demonstrated liver-stage antimalarial activity.