Mice were exposed to phenobarbital or heroin [diacetylmorphine (DAM)] prenatally by feeding the mother phenobarbital on gestation day 9-18; DAM was injected into the mother on gestation days 9-18. At the age of 50 days, mice exposed to phenobarbital or DAM prenatally were examined for long-term biochemical changes in the postsynaptic septohippocampal system as measured by alterations in formation of the second messenger inositol phosphate (i.p.). A significant increase in i.p. formation in response to carbachol was found after prenatal exposure to DAM. An increase in i.p. formation in response to 20 mM KCl alone or in the additional presence of 10 mM carbachol or 1mM physostigmine was found after prenatal exposure to phenobarbital or DAM. In addition, a significant increase in IP formation in response to sodium fluoride was found after prenatal exposure to phenobarbital or DAM. It is suggested that an increase in G-protein activation and in the second messenger formation accompanies the early drug-induced upregulation of the muscarinic receptors found in our previous studies.