Promoter-specific differences in the function of transcription factors play a central role in the regulation of gene expression. We have measured the maximal transcriptional activation potentials of nuclear factor I (NFI) proteins encoded by each of the four identified NFI genes (NFI-A, -B, -C, and -X) by transient transfection in JEG-3 cells using two model NFI-dependent promoters: 1) a simple chimeric promoter containing a single NFI-binding site upstream of the adenovirus major late promoter (NFI-Ad), and 2) the more complex mouse mammary tumor virus long terminal repeat promoter. The relative activation potentials for the NFI isoforms differed between the two promoters, with NFI-X being the strongest activator of NFI-Ad and NFI-B being the strongest activator of the MMTV promoter. To determine if these promoter-specific differences in activation potential were due to the presence of glucocorticoid response elements (GREs), we added GREs upstream of the NFI-binding site in NFI-Ad. NFI-X remains the strongest activator of the GRE containing simple promoter, indicating that differences in relative activation potential are not due solely to the presence of GREs. Since NFI proteins bind to DNA as dimers, we assessed the activation potentials of NFI heterodimers. Here, we show that NFI heterodimers have intermediate activation potentials compared with homodimers, demonstrating one potential mechanism by which different NFI proteins can regulate gene expression.