In this study we show for the first time that pantothenamides also possess antiplasmodial activity. Additionally, we show there to be a labile serum-derived factor common to Albumax II and human serum that specifically antagonizes the antiplasmodial activity of pantothenamides and thereby masks their potency. We demonstrate this factor to be pantetheinase, an enzyme that hydrolyzes pantetheine to form pantothenate and AR-C 102222 cysteamine. This conclusion is based on several findings: that recombinant human pantetheinase also mediates hydrolysis of an antiplasmodial pantothenamide, a finding consistent with earlier reports of secondary amides of pantothenate other than pantetheine serving as pantetheinase substrates ; that the pantothenamide is hydrolyzed in the presence of Albumax II, consistent with Albumax II, like serum, being a source of pantetheinase activity; and that the antiplasmodial potency of the pantothenamide is reduced in the presence of recombinant human pantetheinase and that this attenuating effect is alleviated by incubating the pantetheinasesupplemented culture medium at 37uC. Taken together these results provide strong evidence that in fresh medium, pantetheinase-mediated pantothenamide degradation masks the antiplasmodial potency of pantothenamides. Moreover, we show that in aged culture medium, pantothenamides inhibit growth of P. falciparum with potency unparalleled by antiplasmodial pantothenate analogues identified previously. Five pantothenamides were found to inhibit growth of P. falciparum in aged culture medium with sub-micromolar IC50 values; for one pantothenamide, an IC50 value as low as 20 nM was determined. Furthermore, the finding that the potency of at least one pantothenamide was greater in medium incubated for 80 h than in medium preincubated for 40 h is consistent with there being residual pantetheinase activity in medium pre-incubated for 40 h, and indicates that a maximum pantothenamide potency has not been reached in this medium. To determine the maximum potency of pantothenamides, it will be important to test the pantothenamides under conditions where pantetheinase-mediated degradation cannot take place. This, however, is not a trivial task. Serum albumin is required for growth of P. falciparum and so to achieve strictly pantetheinase-free conditions, an albumin preparation from which all pantetheinase activity has been removed is needed. Whether there is pantetheinase activity associated with preparations of human erythrocytes will also need to be determined. Based on homology searches Andrographolide conducted using the human pantetheinase sequences, the parasite genome appears devoid of putative pantetheinase-encoding genes. This observation, and the finding that the parasite is sensitive to pantothenamide mediated inhibition, suggests that the parasite is incapable of pantetheine hydrolysis.