Acetylcholinesterase (AChE) is transiently expressed by thalamocortical axons in the rat, and staining for this enzyme has been used extensively to study the development of thalamocortical projections. In the present study, patterns of AChE staining were compared in the trigeminal brainstem, thalami and primary somatosensory cortices of perinatal rats, mice, and hamsters. As previously reported, the ventral posteromedial nucleus (VPM) of rats showed dense AChE staining from P-0 at least through P-8. The ventral posterolateral nucleus (VPL) contained heavy AChE staining at least through P-60. In the cortex, there was also dense AChE staining which was organized somatotopically in patches similar to those observed with other methods such as cytochrome oxidase (CO) staining. However, by adulthood, AChE staining revealed a negative image of the CO staining pattern in lamina IV. In the mouse and hamster, there was dense AChE staining inVPL from P-0 through adulthood, but VPM was much less heavily stained for this enzyme. Moreover, the staining in VPL of mice was markedly reduced after transection of axons that travel to the thalamus in the medial lemniscus, suggesting that much of it was contained in these afferent fibers. In the cortices of both perinatal and adult mice and hamsters, AChE staining yielded a negative image of the somatotopically organized patches demonstrable with CO staining. This negative image was apparent by P-2 in the mouse and P-4 in the hamster. These results document a dramatic species difference with respect to the expression of AChE in the thalami and cortices of developing rodents. The differences between the patterns observed in rats vs mice and hamsters probably reflect the fact that cortical AChE in the latter species is not contained in thalamocortical afferents arising from either VPM or VPL.