Depression is a significant public health problem for which currently available medications, if effective, require weeks to months of treatment before patients respond. Previous studies have shown that the G protein responsible for increasing cAMP (G␣ s ) is increasingly localized to lipid rafts in depressed subjects and that chronic antidepressant treatment translocates G␣ s from lipid rafts. Translocation of G␣ s , which shows delayed onset after chronic antidepressant treatment of rats or of C6 glioma cells, tracks with the delayed onset of therapeutic action of antidepressants. Because antidepressants appear to specifically modify G␣ s localized to lipid rafts, we sought to determine whether structurally diverse antidepressants accumulate in lipid rafts. Sustained treatment of C6 glioma cells, which lack 5-hydroxytryptamine transporters, showed marked concentration of several antidepressants in raft fractions, as revealed by increased absorbance and by mass fingerprint. Closely related molecules without antidepressant activity did not concentrate in raft fractions. Thus, at least two classes of antidepressants accumulate in lipid rafts and effect translocation of G␣ s to the non-raft membrane fraction, where it activates the cAMP-signaling cascade. Analysis of the structural determinants of raft localization may both help to explain the hysteresis of antidepressant action and lead to design and development of novel substrates for depression therapeutics.Depression is the leading cause of long term disability in the industrialized world (1). Although depression is a significant health problem in the United States and antidepressants are heavily prescribed (2), the mechanism of action for these drugs is not understood. Further, nearly a third of those treated with these drugs do not achieve remission of their depression (3). Although most of these drugs do interfere with monoamine uptake or catabolism, they exert this effect within hours, even though most of the compounds require weeks before alleviation of symptoms is observed (4). Thus, other targets for antidepressant drugs may exist (4).Chronic antidepressant treatment engages signaling pathways apart from increasing monoamine density in the synaptic cleft. One of these is an increased accumulation of cellular cAMP and sequelae thereof, such as increased cAMP-response element-binding protein (CREB) phosphorylation and increased transcription of cAMP-regulated genes (e.g. BDNF) (5). Moreover, positron emission tomography (PET) evidence suggests that cAMP is diminished throughout the brain of depressed human subjects (6). Thus, it is possible that some antidepressant effects are mediated through induction of the cAMP-generating system, including G␣ s and adenylyl cyclase.Previous studies demonstrated that chronic antidepressant treatment translocates G␣ s from lipid rafts, whereupon it engages in a more facile activation of adenylyl cyclase (7,8). Lipid rafts are regions of the plasma membrane rich in caveolin, cholesterol, sphingolipids, and cytoskeletal an...