Betaine is an important osmolyte and is, compared to other organs, much more abundant in the kidneys where it enters cells in the medulla by the Betaine-GABA-Transporter 1 (BGT1) to balance osmoregulation in the counter current system. In wildtype BGT1 (wt-BGT1)-expressing oocytes, GABA-mediated currents were diminished by preincubation of the oocytes with 100 nM PMA or 5 µM DOG, activators of the protein kinase C (PKC), while application of staurosporine prior to application of DOG restored the response to GABA. Four potential phosphorylation sites on BGT1 were mutated to alanine by site-directed mutagenesis. Three mutants (T235A, S428A, and S564A) evoked GABA currents comparable in magnitude to the currents observed in wt-BGT1-expressing oocytes, whereas GABA currents in T40A were barely detectable. Uptake of [3H]GABA was also determined in HEK293 cells expressing EGFP-tagged BGT1 with the same mutations. T235A, S428A, and S564A showed up-regulation of GABA uptake after hypertonic stress and down-regulation by PMA similar to EGFP-wt-BGT1. In contrast T40A did not respond to either hypertonicity or PMA. Confocal microscopy of the EGFP-BGT1 mutants expressed in MDCK cells revealed that T40A was present in cytoplasm after 24 hours of hypertonic stress while the other mutants and EGFP-wt-BGT1 were in the plasma membrane. All mutants, including T40A, co-migrated with wt-BGT1 on Western blots suggesting that they are full length proteins. T40A, however, cannot be phosphorylated as revealed using a specific anti-phospho antibody and therefore T40 may be important for trafficking and insertion of BGT1 in the plasma membrane.