We investigated the effects of neonatal optic nerve transection on cortical acetylcholinesterase (AChE) activity in hooded rats during postnatal development and following behavioral manipulation after weaning. AChE reaction product was quantified on digitized images of histochemically stained sections in layer IV of primary somatic sensory, primary visual and visual association cortex. Rats with optic nerve transection were compared to sham-operated littermates. In all cortical regions of both types of animal, AChE reaction product was increased to peak 2 weeks after birth and decreased thereafter, reaching adult levels at the end of the third postnatal week. During postnatal development, reaction product in primary visual cortex was lower in rats deprived of retinal input than in sham-operated littermates and the area delineated by reaction product was smaller. However, optic nerve transection did not modify the time course of postnatal development or statistically significantly diminish adult levels of AChE activity. Behavioral manipulations after weaning statistically significantly increased enzyme activity in sham-operated rats in all cortical areas examined. Compared with cage rearing, training in a discrimination task with food reward had a greater impact than environmental enrichment. By contrast, in the rats with optic nerve transection enrichment and training resulted in statistically significantly increased AChE activity only in lateral visual association cortex. Our findings provide evidence for intra- and supramodal influences of the neonatal removal of retinal input on neural activity- and use-dependent modifications of cortical AChE activity. The laminar distribution of the AChE reaction product suggests that the observed changes in AChE activity were mainly related to cholinergic basal forebrain afferents. These afferents may facilitate the stabilization of transient connections between the somatic sensory and the visual pathway.