Unlike the development of acetylcholinesterase (AChE) activity, the postnatal development of the activity of the related enzyme butyrylcholinesterase (BuChE) in the rodent brain has not been investigated in a comprehensive manner. The purpose of the present study was to fill this gap. Development of histochemically visualized BuChE activity followed four distinct stages. Between birth and five postnatal days (P0-P5) BuChE staining of very low intensity was present in nearly all neurons in the forebrain and upper brainstem. Substantial BuChE activity was present in the endothelial cells of blood vessels and the cuboidal cells lining the ventricles. At P6-P10, BuChE neuronal staining of high to moderate intensity emerged in many areas, including certain thalamic nuclei (e.g. anterior group), a number of brainstem nuclei, and darkly stained neurons in the olfactory tubercle/piriform cortex. At P11-P17, the staining which emerged in earlier stages was darker and had expanded to include more neurons. A scattered population of BuChE-positive neurons of moderate to high intensity emerged in the neocortex and amygdala. Importantly, at P17, the very light staining present in all neurons since birth was no longer visible. At P18-P30, the number and staining intensity of cortical neurons displayed a gradual increase while the staining in certain thalamic nuclei was substantially decreased or completely disappeared (e.g. ventral lateral nucleus). A prominent feature of this stage was the emergence of BuChE activity in many fiber tracts. At P30, the adult pattern of staining was attained. The transient presence of BuChE activity of very low intensity in all neurons and of higher intensity in thalamic neurons supports the implied role for this enzyme in neuronal development.