AbstractNuclear factor I (NFI) proteins are DNA‐binding transcription factors that participate in the tissue specific expression of various genes. They are encoded by four different genes (NFI‐A, B, C, and X) each of which generates multiple isoforms by alternative RNA splicing. NFI‐like binding sites have been identified in several genes preferentially expressed in olfactory receptor neurons. Our prior demonstration that NFI binds to these elements led to the hypothesis that NFI is involved in the regulation of these genes. To analyse the role of NFI in the regulation of olfactory neuron gene expression we have performed transient transfection experiments in HEK 293 cells using constructs that place luciferase expression under the control of an olfactory marker protein (OMP)‐promoter fragment containing the NFI binding site. In vitro mutagenesis of this site revealed a negative modulation of luciferase expression by endogenous NFI proteins in HEK 293 cells. In addition, we have used in situ hybridization to analyse the tissue and cellular distribution of the four NFI gene transcripts during pre‐ and postnatal mouse development. We have simultaneously characterized the expression of Pax‐6, and O/E‐1, transcription factors known to regulate the phenotype of olfactory receptor neurons. We demonstrate that all of these transcription factors vary in specific spatio–temporal patterns during the development of the olfactory system. These data on NFI activity, and on transcription factor expression, provide a basis to understand the role of NFI in regulating gene expression in olfactory receptor neurons.