We have examined the effect of mutating the Olf-1 binding motif of the olfactory marker protein (OMP) promoter in determining olfactory neuron-specific gene expression in adult tissues and during embryonic development. The proximal Olf-1 motif located 170 nucleotides upstream of the transcription start site of the OMP gene was mutated to prevent its interaction with the Olf-1 factor in vitro. The wild-type and mutated fragments of the OMP gene extending from -239 to +55 nucleotides relative to the transcription start site were used to direct expression of a lacZ reporter gene in transgenic mice. The transgenic animals were analyzed for cell-specific and developmental expression of the reporter gene. We demonstrate that the mutation that prevents interaction of Olf-1 with its binding site does not alter the temporal and spatial patterns of gene expression in olfactory sensory neurons but does alter the specificity and level of expression in other neuronal populations. These observations are consistent with our demonstration that the mutated Olf-1 site interacts with nuclear proteins present in the central nervous system (CNS).