Toxoplasma gondii invades and proliferates in human umbilical vein endothelial cells (HUVEC) where it resides in a parasitophorous vacuole (PV) preventing lysosomal fusion. To study the intracellular outcome of PV containing tachyzoites of T. gondii during interaction with IFN-γ–activated HUVEC, a quantitative analysis of the T. gondii infection and multiplication was assayed. The quantification of PVs' fusion with lysosomes, ultrastructural examination of phagosome–lysosome fusion, and the localization of NAD(P)H-oxidase activity were also investigated. HUVEC activated with IFN-γ inhibited T. gondii infection and multiplication by 67.5% and 91.0%, respectively. After 4 hr of infection, 10.2% of IFN-γ–activated HUVEC exhibited phagosome–lysosome fusion assayed by fluorescence microscopy, which was also observed at the ultrastructural level. Furthermore, the enzyme NAD(P)H-oxidase present at the plasma membrane of activated HUVEC was internalized together with the parasite in 38.0% of the cells. In addition, colocalization of colloidal gold particles and reaction product of NAD(P)H-oxidase in the PV of some activated HUVEC was observed. These results suggest that NAD(P)H-oxidase may participate in a mechanism by which IFN-γ–activated HUVEC inhibit T. gondii multiplication.