We have used in situ hybridization to characterize the ontogeny of alpha3 and beta4 nicotinic acetylcholine receptor (nAChR) subunit mRNA expression in rat brain. Transcripts for both subunits were detected in embryonic brain, although overlapping expression of alpha3 mRNA was only evident in areas of strong beta4 mRNA expression, including the medial habenula, locus coeruleus, the cerebellar primordium, and several motor and sensory brainstem nuclei. During the perinatal period, the independent expression of alpha3 mRNA declined, and greater correspondence in the temporal and spatial expression of alpha3 and beta4 subunit mRNAs emerged. In general, beta4 mRNA expression preceded that of alpha3 mRNA by 1 to 2 days. Overlapping expression patterns were transiently detected in the caudate putamen, basal forebrain, frontal and visual cortices, and in the CA3 field of hippocampus. Codistribution that lasted throughout development and into adulthood was noted in a number of brain areas, including the retrosplenial cortex, subiculum, medial habenula, interpeduncular nucleus, locus coeruleus, and brainstem motor nuclei. In many of these regions, alpha5 subunit mRNA was also expressed. Colocalization of alpha3 and beta4 mRNAs with choline acetyltransferase mRNA was detected in cholinergic neurons of the brainstem motor nuclei, nucleus ambiguus, dorsal motor nucleus of the vagus, motor trigeminal nucleus, and facial nucleus, but not in most forebrain cholinergic cells. The extensive correspondence in temporal and spatial distribution of alpha3 and beta4 mRNAs throughout postnatal brain development suggests that these subunits may be coordinately regulated and may form functional neuronal nAChRs with significant developmental roles.