The determinants of in vivo target site selectivity by transcription factors are poorly understood. To find targets for the developmentally regulated transcription factor TBX2, we generated stable transfectants of human embryonic kidney cells (293) that express a TBX2-ecdysone receptor (EcR) chimeric protein. While constitutive expression of TBX2 is toxic to 293 cells, clones expressing TBX2EcR are viable in the absence of an EcR ligand. Using cDNA arrays and quantitative PCR, we discovered nine genes whose expression was increased, but no genes whose expression was reduced, following 24 h of induction with Ponasterone A (PonA), a ligand for EcR. Since TBX2 was reported previously to be a transcriptional repressor, we also generated cell lines expressing a TBX2VP16EcR protein which we showed was a potent conditional transcriptional activator in transient transfection assays. Treatment of these cells with PonA induced the expression of five genes, none of which were affected in TBX2EcR-expressing cells. This discordance between TBX2- and TBX2VP16-regulated genes strongly suggests that specific transactivation domains can be a major determinant of gene target site selectivity by transcription factors that possess the same DNA-binding domain.