The human Anion exchanger 1 (AE1, band 3, SLC4A1) is highly expressed in erythrocytes and ∝-intercalated cells of the kidney. The kidney AE1 (kAE1) is a truncated version of AE1 found in the basolateral membrane and its correct localisation and function is essential for renal acid secretion. However, despite its importance in whole body pH homeostasis surprisingly little is known about the trafficking and regulation of human kAE1. We have developed a polarisable cell model system expressing human kAE1 in MDCKI cells (MDCKI-kAE1 cells) to investigate the regulation of the trafficking of kAE1. We have already shown, using this model system, that the C-terminal tyrosine residue (Y904) is critical for basolateral localisation in polarised cells and that the N-terminus of kAE1 is also essential for polarized trafficking. We have now utilized mutagenesis to further investigate the regions of the N-terminus important for kAE1 trafficking. In particular, we have found that the Y359A mutant is mistargeted to the apical membrane. Therefore, kAE1 has a requirement for the presence of both Y359 in the N-terminus and Y904 in the C-terminus for correct trafficking. Tyrosine phosphorylation can influence the localisation of proteins by regulating interactions with cellular targeting and/or internalisation machinery. Both Y359 and Y904 are phosphorylated in erythrocyte AE1. We show by cellular imaging and western blotting techniques using novel phosphotyrosine-specific antibodies raised against AE1 phosY359 and AE1 phosY904, that both Y359 and Y904 are phosphorylated in pervanadate treated MDCK-kAE1 cells. This phosphorylation was sensitive to Src kinase inhibitors. Therefore since both Y359 and Y904 are critical for basolateral targeting, we propose that reversible phosphorylation provides a means of regulating kAE1 trafficking to the basolateral membrane. We suggest that intercalated cells can regulate the levels and subcellular distribution of kAE1 in response to cellular stimuli and cues (such as acidosis or alkalosis) by reversible phosphorylation of Y359 and Y359. We are now exploring a range of stimuli to determine the physiological signals that influence kAE1 phosphorylation and their effects on basolateral localisation.