Olfactory receptor neurons are responsible for the detection and signal transduction of odor ligands. Several genes associated with this activity are preferentially or exclusively expressed in these neurons. Among these genes are those coding for olfactory receptors, adenylyl cyclase type III, the cyclic nucleotide gated olfactory channel 1 (OcNC-1), Galpha(olf) and the olfactory marker protein (OMP). Promoter analyses of these genes identified a binding site for the new transcription factor family O/E whose initial member, Olf-1, is abundantly expressed in olfactory neurons. We report here that the proximal promoters of three of these genes, that are selectively expressed in olfactory neurons, each contains a functional NFI binding site and that the sites have different affinities for NFI proteins indicating a regulatory role for NFI proteins in olfactory gene expression. We further demonstrate, by cloning, that all four NFI genes are expressed in the olfactory nasal mucosa. Analysis by in situ hybridization illustrates that at least three of these gene products are expressed in the neuroepithelium in which the olfactory neurons reside. NFI proteins are capable of functioning as positive or negative regulators of transcription depending on the tissue, cell-type, age, and gene in question. These multivalent functions of NFI could be achieved by temporally and spatially regulated expression of distinct subsets of NFI isoforms. It now remains to characterize the tissue and cell specific patterns of expression of distinct NFI transcription factors during ontogeny and their roles in regulating gene expression.