IPC (inositol phosphorylceramide) synthase is an enzyme essential for fungal viability, and it is the target of potent antifungal compounds such as rustmicin and aureobasidin A. Similar to fungi and some other lower eukaryotes, the protozoan parasite Trypanosoma cruzi is capable of synthesizing free or protein-linked glycoinositolphospholipids containing IPC. As a first step towards understanding the importance and mechanism of IPC synthesis in T. cruzi, we investigated the effects of rustmicin and aureobasidin A on the proliferation of different life-cycle stages of the parasite. The compounds did not interfere with the axenic growth of epimastigotes, but aureobasidin A decreased the release of trypomastigotes from infected murine peritoneal macrophages and the number of intracellular amastigotes in a dose-dependent manner. We have demonstrated for the first time that all forms of T. cruzi express an IPC synthase activity that is capable of transferring inositol phosphate from phosphatidylinositol to the C-1 hydroxy group of C6-NBD-cer {6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-amino]hexanoylceramide} to form inositol phosphoryl-C6-NBD-cer, which was purified and characterized by its chromatographic behaviour on TLC and HPLC, sensitivity to phosphatidylinositol-specific phospholipase C and resistance to mild alkaline hydrolysis. Unlike the Saccharomyces cerevisiae IPC synthase, the T. cruzi enzyme is stimulated by Triton X-100 but not by bivalent cations, CHAPS or fatty-acid-free BSA, and it is not inhibited by rustmicin or aureobasidin A, or the two in combination. Further studies showed that aureobasidin A has effects on macrophages independent of the infecting T. cruzi cells. These results suggest that T. cruzi synthesizes its own IPC, but by a mechanism that is not affected by rustmicin and aureobasidin A.