Acetylcholinesterase (AChE) is transiently expressed in several immature axon systems. Its presence in developing thalamocortical afferents has led to the use of enzyme histochemistry to visualize this axon system in rats. Because of the spatiotemporal distribution of the enzyme in the rat neocortex, it has been suggested that AChE plays a role in the establishment of thalamocortical connectivity. We show here that AChE is distributed in a pattern that is markedly different in SI cortex of rats as compared to that of mice and hamsters. In rat pups, AChE-rich patches are distributed in a vibrissa-related array in the SI cortex soon after birth, whereas regions of cortex that lie between individual patches, and between rows of patches, are impoverished in the enzyme. In contrast, sections through flattened cortices from PND3 and older mice and hamsters reveal lightly stained AChE-positive spots in the center of barrel cores, while barrel walls remain devoid of AChE; septae that divide individual barrels are densely enzyme positive. Differences in laminar localization of the enzyme for all three species are also visible. In the thalamus of postnatal rats, both the ventral posterior medial (VPM) and ventral posterior lateral (VPL) nuclei express AChE, correlating with the presence of enzyme-containing patches throughout the barrelfield cortex. In the other two rodents, however, the enzyme is present in VPL but not in VPM, despite the fact that in these species the cortical barrels associated with both thalamic nuclei have very little of the enzyme. Thus, the relationship between the distribution of AChE in nuclei of the thalamic ventrobasal complex and the presence of AChE in the terminals of their cortical axons in the barrelfield is not consistent across different rodent species. Our results call for caution in the use of AChE histochemistry as a universal marker for immature thalamocortical axons, and challenge the generality of currently hypothesized roles for this transiently expressed enzyme during the development of the rodent thalamocortical projection.