The tritiated specific antagonist [3H]GR 113808 was used to study the brain regional distribution of 5-HT4 binding sites in rat during development. In adult brain, high densities of binding sites were found in the limbic system (islands of Calleja, olfactory tubercle, fundus striati, ventral pallidum, septum, hippocampus and amygdala) and in the striato-nigro-tectal pathway. Only low densities of binding sites were found in the prenatal brain, except in the brainstem. Marked density increases were observed during the second and third postnatal week in most forebrain regions. This ontogenic pattern indicates that 5-HT4 receptors probably exert a minor role in developmental processes, while, in the adult, the expression of 5-HT4 receptors in limbic areas strongly suggests a role for these receptors in emotional functions. The synchronous appearance of 5-HT4 receptors and cholinergic markers also indicates that these receptors might control acetylcholine release. This hypothesis has been confirmed by functional evidence and further supports the view that 5-HT4 drugs could improve cognitive functions in diseases with impaired cholinergic transmission (e.g., Alzheimer's disease).