P2X4 receptors are a subtype of ATP-gated cation channel and are composed of two transmembrane domains, an extracellular ligand binding site and intracellular N- and C-termini. Peripheral nerve damage has been demonstrated to cause upregulation of the receptor resulting in tactile allodynia, which was reduced following adminstration of P2X4 anti-sense oligodeoxynucleotides (Tsuda et al. 2003). When heterologously expressed in neurones and human embryonic kidney (HEK-293) cells P2X4 receptors undergo constitutive endocytosis through an interaction with a non-canonical tyrosine based motif and adaptor protein-2 (Bobanovic et al. 2002; Royle et al. 2002; Royle et al. 2005). Recent work in primary macrophages has shown that P2X4 receptors are predominantly located within lysosomes, where they retain their function and can be delivered to phagosomes and the plasma membrane (unpublished observations, Qureshi & Murrell-Lagnado). We show by immunocytochemistry in normal rat kidney (NRK) cells that targeting of rat P2X4 receptor to lysosomes involves a dileucine type motif in the N-terminus in addition to previously identified tyrosine based motifs in the C-terminus (Royle et al. 2002). Mutation of these motifs results in increased surface expression of the receptor. When both the tyrosine and dileucine motifs are mutated there is reduced lysosomal localisation of the receptor. Furthermore, Western blot analysis demonstrates that the P2X4 receptor remains stable within the proteolytic environment of the lysosomes. Seven potential N-linked glycosylation sites have been identified on the extracellular loop of P2X4 and we provide evidence that at least six are glycosylated. Removal of these glycans in vivo with endoglycosidase H results in the rapid degradation of the receptor. Our results show that P2X4 receptors are targeted to lysosomes by dileucine- and tyrosine-based motifs, where they are protected from degradation by N-linked glycosylation.