To follow on a recent quantitative study of the developing cholinergic (ACh) innervation in rat neocortex, axon varicosities identified by electron microscopic immunocytochemistry with a highly sensitive antibody against choline acetyltransferase (ChAT) were examined in the primary somatosensory area (Par1) of rats at postnatal ages (days) P8, P16, and P32. As visualized and measured in single thin sections, and compared with those of unlabeled varicosities selected at random in the same photomicrographs, the ChAT-immunostained profiles displayed intrinsic and relational features very similar to those previously described in the same cortical area of adult rat (Umbriaco et al.  J. Comp. Neurol. 348:351–373). At the three postnatal ages, the immunoreactive profiles were comparable in shape, size, and vesicular content in all cortical layers, but showed an increasing frequency of mitochondria with age, reaching 44% at P32. Synaptic junctions were observed on 6.3 to 8.7% of these sectional profiles, indicating an average synaptic incidence of 17% for whole varicosities, again comparable to that in the adult (14%). As in adult, the junctions made by the rare synaptic ChAT-immunostained varicosities were always single, usually symmetrical, and more frequently found on dendritic branches than spines. Thus, cortical ACh varicosities displayed intrinsic and relational features similar to adult ones as soon as this innervation was installed, suggesting that a diffuse mode of transmission and ambient level of ACh could play a major role in the diverse effects of this neuromodulator during cortical development. J. Comp. Neurol. 443:250–258, 2002. © 2002 Wiley-Liss, Inc.