Bioluminescent reporter assays have been widely used to study the effect ofToxoplasma gondiion host gene expression. In the present study we extend these studies by engineering novel reporter cell lines containing a gamma activated sequence (GAS) element driving Firefly luciferase (FLUC). In RAW264.7 macrophages,T. gondiitype I strain (GT1) infection blocked interferon-gamma (IFN-γ) induced FLUC activity to a significantly greater extent than infection by type II (ME49) and type III (CTG) strains. Quantitative trait locus (QTL) analysis of progeny from a prior genetic cross identified a genomic region on chromosome XII that correlated with the observed strain-dependent phenotype. This QTL region contains two isoforms of theT. gondiienzyme Nucleoside Triphosphate Hydrolase (NTPase) that were the prime candidates for mediating the observed strain-specific effect. Using reverse genetic analysis we show that deletion of NTPase I from a type I strain (RH) background restored the higher luciferase levels seen in the type II (ME49) strain. Rather than an effect on IFN-γ-dependent transcription, our data suggest that NTPase I was responsible for the strain-dependent difference in FLUC activity due to hydrolysis of ATP. We further show that NTPase I and II were not essential for tachyzoite growthin vitroor virulence in mice. Our study reveals that whileT. gondiiNTPases are not essential for immune evasion, they can affect ATP-dependent reporters. Importantly, this limitation was overcome using an ATP independent Gaussia luciferase (GLUC), which provides a more appropriate reporter for use withT. gondiiinfection studies.