The Plasmodium falciparum malarial parasite genome appears to encode one and only one phosphatidylinositol 3'-kinase (PI3K) and sequence analysis suggests that the enzyme is a "class III" or "Vps34" type PI3K. PfVps34 has generated excitement as a possible druggable target and potentially a key target of artemisinin-based antimalarials. In this study, we optimize the PfVps34 gene for heterologous expression in yeast, purify the protein to homogeneity, use a recently validated quantitative assay for phosphatidylinositol 3'-phosphate (PI3P) production from phosphatidylinositol (PI) (Hassett et al., previous paper, this issue) to quantify activity and drug inhibition of that activity, and investigate the importance of key residues in the enzyme's catalytic and "Nlobe" domains. Data suggest that PfVps34 is indeed inhibited by artemisinin and related drugs, but only under conditions that cleave the drugs' endoperoxide bridge to generate reactive alkylating agents.