Here we investigate differences in the temporal and spatial patterning, and subunit interactions of two of the major Na⁺ channel α‐subunit isoforms in mammalian brain, the type I and type II Na⁺ channels. By using subtype‐specific antibodies, we find that both isoforms are abundant in adult rat brain, where both interact with the covalently bound β2 auxiliary subunit. Immunoblot analysis reveals complementary levels of type I and type II in different brain regions, with the highest levels of type I in brainstem, cortex, substantia nigra, and caudate, where it is found predominantly on the soma of neurons, and the highest levels of type II in globus pallidus, hippocampus and thalamus, where it is preferentially localized to axons. Developmentally, type I Na⁺ channel polypeptide expression in brain increases dramatically during the third postnatal week, peaks at the end of the first postnatal month, and then decreases such that adult levels are ≈ 50% of those at peak. Type II Na⁺ channel polypeptide expression in brain also undergoes large increases in the third postnatal week, but levels continue to increase such that peak expression levels are maintained in adult animals. Type I Na⁺ channels are found associated with the auxiliary β2 subunit at all ages, whereas free type II Na⁺ channels exist during the first two postnatal weeks. Thus, although expression of these two Na⁺ channel α subunits in heterologous systems yields currents with very similar electrophysiological and pharmacological properties, their distinct spatial and temporal patterning, and association with auxiliary subunits in brain, suggest that they perform distinct, nonoverlapping functions in situ. J. Comp. Neurol. 412:342–352, 1999. © 1999 Wiley‐Liss, Inc.