The effects of early postnatal (PD 8) intracerebroventricular injection of ethylcholine mustard aziridinium ion (AF64A) on development of open-field and cognitive behaviors and cholinergic markers in several brain areas were examined in the rat. The cholinotoxin was bilaterally administered in a dose range of 0.25 to 2.0 nmol. In the open-field tests, the cholinergic lesion caused a dose-dependent increase in activity at 20 days of age, while it resulted in lengthened latency to initiate exploration and decreased rearing activity at adulthood. Hole-board spatial learning was severely inhibited in adult age. The biochemical activity of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in the hippocampus was markedly decreased in a dose-dependent manner, but was unchanged in the neocortex and striatum. Histochemical staining of AChE-positive fibers revealed a severe cholinergic denervation of the granular and pyramidal cell layers of the hippocampus. The results showed that a selective cholinergic deafferentation of the hippocampus at a critical stage of development leads to long-lasting abnormal open-field and spatial learning behaviors.