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 (Gαs) is increasingly localized to lipid rafts in depressed subjects and that chronic antidepressant treatment translocates Gαs from lipid rafts. Translocation of Gαs, 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 Gαs 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 Gαs 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.