We have investigated the subcellular accumulation and transport of camptothecin (CPT), a monoterpene indole alkaloid, in hairy roots of Ophiorrhiza pumila. This hairy root produces high amounts of CPT and excretes it into the culture medium. When the hairy roots were exposed to UV radiation, autofluorescence emitted from CPT showed subcellular localization of CPT in the vacuole. Treatment with several inhibitors suggested that CPT excretion is a transporter-independent passive transport controlled by the concentration gradient of the compound. Interestingly, the hairy roots treated with brefeldin A, a vesicle transport inhibitor, showed increased CPT excretion. This could be explained by an increased transport rate of CPT from the endoplasmic reticulum (ER) to the cytoplasm when transport of CPT to the vacuole is blocked. The much higher concentration of CPT in the cytoplasm resulted in the increased excretion rate. This result indicates that CPT is biosynthesized at the ER and transported to accumulate in the vacuole by the same machinery that is used for vacuolar protein sorting. How O. pumila is insensitive to CPT is discussed.