F5 was first identified as an mRNA expressed by activated but not resting T-lymphocytes. Subsequent studies suggested that it also is selectively expressed by mature neurons. Although the F5 protein coding sequence is highly conserved, the function of the F5-encoded protein is unknown. The present studies were undertaken to define the anatomic distribution, cellular specificity, and developmental pattern of F5 mRNA expression in the mouse nervous system, addressing specifically the question of whether the expression pattern of F5 corresponds to that of known ligand-receptor or signal-transduction systems. The use of a nonradioactive in situ hybridization method and paraffin-embedded sections provided excellent morphological preservation and a high degree of cellular resolution. F5 mRNA was detected in the central nervous system, peripheral nervous system, and retina in cells having the location and morphological features of neurons. Combined in situ hybridization histochemistry for F5 mRNA and immunofluorescence staining for cell-specific markers confirmed that neurons expressed F5 mRNA but astrocytes did not. The neuronal expression of F5 mRNA had two interesting features. First, the level of expression appeared to correlate directly with the size of the neuronal perikarya, the length of the axonal projection, or the extent of dendritic arborization. Second, F5 mRNA appeared late in post-natal development. These observations are of interest because of preliminary data suggesting that F5 may function as a substrate for protein kinase C, which demonstrates a similar expression pattern in the nervous system.