Cannabinoid receptors are found in moderate density throughout the cerebral cortex. The anterior cingulate cortex (ACC) is of particular interest due its high level of cannabinoid receptors and role in behaviors known to be modulated by cannabinoids. These studies were conducted to determine the cellular localization of cannabinoid receptors and to compare the level of cannabinoid receptor binding with receptor-mediated G-protein activity in the rat ACC. Either ibotenic acid or undercut lesions were made in ACC, and brains were processed for [3H]WIN 55,212-2 and WIN 55,212-2-stimulated [35S]GTPgammaS autoradiography. Both cannabinoid receptors and receptor-activated G-proteins were highest in laminae I and VI of ACC in control tissue. Although similar levels of receptor binding were found in these laminae, significantly higher levels of receptor-activated G-proteins were found in lamina VI. Ibotenic acid lesions that destroyed ACC neurons decreased [3H]WIN 55,212-2 binding by 60-70% and eliminated WIN 55,212-2-stimulated [35S]GTPgammaS binding. In contrast, deafferentation of the ACC with undercut lesions had no significant effect on cannabinoid receptor binding or G-protein activation. These results indicate that cannabinoid receptors in laminae I and VI of the ACC are located on somatodendritic elements or axons intrinsic to the ACC. In addition, differences in the relative levels of cannabinoid binding sites and activated G-proteins between cortical laminae indicate that the efficiency of cannabinoid receptors for G-protein activation may vary within a specific brain region.