Whether the mechanism that Agmatine sulfate ultimately results in bacteriostasis is inhibition of CoA biosynthesis, fatty acid biosynthesis or another CoA-utilizing process, or a combination of the above, remains to be resolved. In this study, the effect of a series of pantothenamides on the growth of erythrocytic stage P. falciparum parasites was investigated. We show for the first time that under standard in vitro culture conditions pantothenamides inhibit parasite growth, albeit with modest potency. Serendipitously, however, we discovered that the antiplasmodial potency of pantothenamides is enhanced considerably when the parasite culture medium used for growth assays is pre-incubated at 37uC for a prolonged period. Consequently, sub-micromolar concentrations of pantothenamides that have no effect in freshly prepared medium inhibit parasite growth effectively in the preincubated medium. We present evidence that links this finding to the presence in parasite culture medium of pantetheinase activity, the activity of an enzyme that catalyzes the hydrolysis of pantetheine to pantothenate and cysteamine. In animals, pantetheinase activity is typically linked with the Vanin proteins, soluble or membrane bound proteins that belong to the nitrilase superfamily, the members of which share an invariant Glu-Lys-Cys Endoxifen catalytic triad. We show, using an in vitro primary amine detection assay, that a pantothenamide selected from the series tested here is hydrolyzed in the presence of Albumax II, demonstrating Albumax II to be a source of pantetheinase activity. Furthermore, we show that recombinant human pantetheinase reduces the antiplasmodial potency of the pantothenamide in the pre-incubated medium in vitro, and, that the attenuating effect of the pantetheinase is alleviated by incubation of the pantetheinase-supplemented medium at 37uC. Together these data are consistent with pantetheinase-mediated pantothenamide degradation occurring in medium freshly supplemented with Albumax II or serum under in vitro culture conditions, lowering the effective pantothenamide concentration, and thereby masking the sub-micromolar antiplasmodial potency of pantothenamides. Importantly, we demonstrate that the potent antiplasmodial effect of the pantothenamides in the medium pre-incubated at 37uC is alleviated with pantothenate, and therefore results specifically from inhibition of pantothenate and/or CoA utilization. We also show that all of the pantothenamides in this series inhibit P. falciparum PanK-catalysed pantothenate phosphorylation. The data presented here provide additional validation of pantothenate and CoA utilization as potential antiplasmodial drug targets. The data presented thus far are consistent with there being a heat-labile component in Albumax-complete RPMI that antagonizes the activity of pantothenamides. In an attempt to identify such a component, the activity of a selected pantothenamide in aged Albumax-complete RPMI supplemented immediately prior to the assay with various components of Albumax-complete RPMI, was investigated.