We investigated the transduction mechanisms mediating formation of the endogenous cannabinoid (endocannabinoid) lipids, anandamide (arachidonylethanolamide) and 2-arachidonylglycerol, in primary cultures of rat cortical neurons. Unstimulated neurons contained 0.3 +/- 0.1 pmol of anandamide and 16.5 +/- 3.3 pmol of 2-arachidonylglycerol per mg of protein, as determined by gas chromatography/mass spectrometry. Ca(2+) entry into the neurons via activated glutamate N-methyl-D-aspartate (NMDA) receptors increased 2-arachidonylglycerol levels approximately three times, but had no effect on anandamide levels. By contrast, anandamide formation was stimulated five times by simultaneous activation of NMDA and acetylcholine receptors. Alone, acetylcholine receptor activation had no effect on anandamide or 2-arachidonylglycerol levels. The formation of fatty acid ethanolamides that do not activate cannabinoid receptors, including palmitylethanolamide and oleylethanolamide, was stimulated by coactivation of NMDA and acetylcholine receptors. Pharmacological experiments suggest that the cholinergic contribution to anandamide formation was mediated by alpha7 nicotinic receptors (antagonized by methyllycaconitine), whereas the contribution to palmitylethanolamide and oleylethanolamide formation was mediated by muscarinic receptors (antagonized by atropine). These findings indicate that cortical neurons produce anandamide and 2-arachidonylglycerol in a receptor-dependent manner, and that brain neurons may generate different endocannabinoid lipids depending on their complement of neurotransmitter receptors.