We examined the mechanisms involved in protein kinase C (PKC)‐dependent down‐regulation of dopamine transporter (DAT) activity and cell surface expression by treating heterologously expressing cells with the clathrin‐mediated endocytosis inhibitor concanavalin A (Con A) or the cholesterol depleter/membrane raft disrupter methyl‐β‐cyclodextrin (MβC) prior to treatment with the PKC activator phorbol 12‐myristate, 13‐acetate (PMA). Con A blocked PMA‐induced surface reductions of DAT but only partially inhibited down‐regulation, while MβC partially blocked down‐regulation but did not inhibit loss of cell surface DAT, demonstrating that PKC‐induced DAT down‐regulation occurs by a combination of trafficking and non‐trafficking processes. Using density‐gradient centrifugation, we found that DATs are distributed approximately equally between Triton‐insoluble, cholesterol‐rich membrane rafts and Triton‐soluble non‐raft membranes. DATs in both populations are present at the cell surface and are active for dopamine and cocaine binding. PMA‐induced loss of cell surface DAT occurred only from non‐raft populations, demonstrating that non‐raft DATs are regulated by trafficking events and indicating the likelihood that the cholesterol‐dependent non‐trafficking regulatory mechanism occurs in rafts. PMA did not affect the DAT raft‐non‐raft distribution but stimulated the phosphorylation of DAT to a substantially greater level in rafts than non‐rafts. These findings reveal a previously unknown role for cholesterol in DAT function and demonstrate the presence of distinct subcellular DAT populations that possess multiple regulatory differences that may impact dopaminergic neurotransmission.