Histochemical mapping of AChE activity in the chick diencephalon shows differential staining of several subregions within the nucleus rotundus. The topography and extent of these subdivisions were studied in transverse, horizontal and sagittal sections. A correlation with rotundic hodologic subdivisions reported in the literature is feasible, whereas several other chemoarchitectonic or functional markers show a homogeneous distribution throughout the n. rotundus. Moreover, cholinergic markers do not detect cholinergic afferents within the rotundic neuropile. Late embryonic appearance of the AChE heterogeneity suggests a modulation of neuropile AChE levels subsequent to synaptogenetic adjustment of differential hodology.