Abstract Mice were exposed to phenobarbital(PhB) prenatally and neonatally. Prenatal exposure was accomplished by feeding the mother PhB (3 g/kg milled food) on gestation days 9–18. Neonatal exposure was accomplished by daily injections of 50 mg/kg sodium PhB directly to the pups on days 2–21. Long-term biochemical alterations in the pre- and postsynaptic septohippocampal system, as well as related behavioral deficits, were assessed in the treated animals. Significant increase in B max values for binding of [ 3H]QNB to muscarinic cholinergic receptors was obtained on both ages 22 and 50 in prenatally (40–90%, respectively, p < 0.001) and neonatally exposed (58–89%, p < 0.001) mice whereas K d remained normal. Similarly, a significant increase of inositol phosphate (IP) formation in response to carbachol was found after both prenatal and neonatal exposure to PhB ( p < 0.05). No alterations in choline acetyltransferase (ChAT) activity were observed in the prenatally or neonatally treated animals. The early exposed mice showed deficits in the performance in Morris water maze, a behavior related to the septohippocampal pathway. The results suggest that early exposure to PhB induces alterations in postsynaptic components of the hippocampal cholinergic system and concomitantly to impairment in hippocampus-related behavior.