Vacuole biogenesis, integrity and function depend on the targeting of membrane proteins to this organelle. The transport of membrane proteins to the vacuole is thought to occur by vesicle trafficking from the endoplasmic reticulum after translocation to the ER membrane. Two pathways have been proposed for the targeting of tonoplast proteins through the endomembrane system, one that is Golgi-dependent and another that is Golgi-independent. The Golgi-dependent pathway was described for a chimeric LEE011 protein containing the transmembrane domain and C-terminus of pea BP-80. In tobacco RAD001 protoplasts this protein was targeted via a Brefeldin A – sensitive pathway towards a pre-vacuolar compartment. BFA is an inhibitor of Golgi-dependent traffic because it inhibits COPI coat formation and retrograde trafficking from the Golgi to the ER. Consistent with evidence for Golgi post-translational modifications, the BP-80 fusion protein contained Asn-linked glycans. Recent evidence suggests that there may be two Golgi-dependent targeting pathways that differ by their dependence on the adaptor protein complex AP3. Evidence for a Golgi-independent pathway was first obtained in tobacco protoplasts, when Wortmannin and BFA did not inhibit the delivery of a-TIP to the vacuole. Later, it was shown that in tobacco protoplasts the C terminus of bean a-TIP was sufficient to prevent a reporter protein from entering the Golgi in its route to the vacuole. In Arabidopsis protoplasts, the trafficking of a HA-TIP3;1 is insensitive to BFA treatment, the co-expression of the dominant negative form AtRab1, or the overexpression of Atsec23, all of which inhibit ER-Golgi traffic. In addition, Arabidopsis TIP3;1-YFP targeting was dependent on COPII and this cargo was mis-targeted when mutant forms of Rha1, Ara6 and Rab7 were transiently overexpressed in tobacco leaf epidermal cells. Similarly, the trafficking of the rice Two-Pore K + b channel is also BFA-insensitive. While there is some evidence for Golgi-independent trafficking of tonoplast proteins in Arabidopsis and tobacco, all the data thus far have come from transient expression studies and, in most cases, heterologous systems. Maintenance of vacuolar membrane integrity is essential for plant growth and development, and yet little is known about the mechanisms regulating the trafficking of membrane proteins to the vacuole. Unlike an extensive record for trafficking of soluble vacuolar proteins, only a few endomembrane components, including the SNARE protein SYP21, and three Rab proteins, Rha1, Ara6 and Rab7, have been implicated in tonoplast membrane trafficking.