Postnatal X-irradiation of the rat hippocampus results in a marked reduction in the number of the postnatally developing granular neurons in the dentate gyrus and also caused a marked increase in the specific activity of acetylcholinesterase (AChE) and choline acetyltransferase (CAT) and a slight but consistent increase in the activity per whole hippocampus of AChE. The effect of irradiation on the granular neurons and on the cholinergic enzymes was found to be dose and age dependent. Drastic increase in specific enzymatic activities is also observed in the irradiated cerebellum whose granular neurons differentiate postnatally and to a lesser extent in the cerebral cortex in which cell formation is accomplished prior to birth. Staining for AChE activity revealed enhanced staining in the molecular layer and the hilar zone of the irradiated dentate gyrus, and in the striatum lucidum of area CA3 which corresponds to the projection area of the mossy fibers. Enhanced staining in area CA1 and subiculum was noticed especially in the supra- and infrapyramidal layers. Biochemical analysis demonstrated that AChE and CAT activities were 140-180% higher in the subareas of the irradiated vs non-irradiated hippocampus. The development and distribution of the postsynaptic muscarinic receptors in the irradiated hippocampus by [3H]quinuclidinyl benzilate (QNB)-binding were also studied. It was found that the elimination of the postnatally formed neurons does not appear to change the developmental pattern of the [3H]QNB-binding sites but reduced receptor level to about 75% of control to adulthood. Measurements of the [3H]QNB-binding in the subareas within the hippocampus revealed marked reduction in the specific [3H]QNB-binding in the molecular layer of the dentate gyrus but not in other subareas. However, the reduction in [3H]QNB-binding sites in the dentate is not as drastic as the reduction in the number of granular neurons. It is suggested that muscarinic sites may be located on early formed neurons, non-cholinergic afferents, or glial elements in this area.