Synergistic interaction between atovaquone and proguanil has been suggested as the reason for the effectiveness of Malarone. The pharmacodynamic interactions among atovaquone, proguanil and its metabolite cycloguanil were investigated in 4 Plasmodium falciparum parasite strains by culture assays in vitro. The response parameters were determined and 2 statistical methods, log-concentration/response probit method and sum of fractional inhibitory concentrations (sigmaFIC) method, were used to analyse the experimental data. Within therapeutically relevant concentration ratios, the combination of atovaquone and proguanil showed mean sigmaFICs of 0.37 at EC50 (50% effective concentrations) and 0.13 at EC90, indicating high synergism. The combination of atovaquone and cycloguanil yielded corresponding mean sigmaFICs of 3.70 and 2.11, indicating antagonism. The EC50 and EC90 values for proguanil alone were not influenced by RPMI-1640 medium with low concentrations of paraaminobenzoic acid and folic acid (LPLF culture medium), whereas the EC50 and EC90 values for cycloguanil were more than 10 times lower in LPLF medium than in normal RPMI-1640 medium. This confirms the hypothesis that proguanil may act on another target than dihydrofolate reductase. We conclude that the effectiveness of Malarone is due to the synergism between atovaquone and proguanil and may not require the presence of cycloguanil.