Previous studies demonstrated that Gsalpha migrates from a Triton X-100 (TTX-100) insoluble membrane domain to a TTX-100 soluble membrane domain in response to chronic treatment with the antidepressants desipramine and fluoxetine. Antidepressant treatment also causes a Gsalpha redistribution in cells as seen by confocal microscopy. The current studies have focused on examining the possibility that the association between Gsalpha and the plasma membrane and/or cytoskeleton is altered in response to antidepressant treatment, and that this is relevant to both Gsalpha redistribution and the increased coupling between Gsalpha and adenylyl cyclase seen after chronic antidepressant treatment. Chronic treatment of C6 cells with two fuctionally and structurally distinct antidepressants, desipramine and fluoxetine, decreased the Gsalpha content of TTX-100 insoluble membrane domains by as much as 60%, while the inactive fluoxetine analog LY368514 had no effect. Disruption of these membrane domains with the cholesterol chelator methyl-beta-cyclodextrin altered the localization of many proteins involved in the cAMP signaling cascade, but only Gsalpha localization was altered by antidepressant treatment. In addition, microtubule disruption with colchicine elicited the movement of Gsalpha out of detergent-resistant membrane domains in a manner identical to that seen with antidepressant treatment. The data presented here further substantiate the role of Gsalpha as a major player in antidepressant-induced modification of neuronal signaling and also raise the possibility that an interaction between Gsalpha and the cytoskeleton is involved in this process.