Introduction: Sphingosine-1-Phosphate (S1P) is a primary modulator of resistance artery tone. Its bioavailability is controlled by a rheostat between sphingosine kinase 1 (Sk1) and intracellular S1P phosphohydrolase 1 (SPP1). We have found that the transport by the Cystic Fibrosis Transmembrance Conductance Regulator (CFTR), a chloride channel, is the rate-limiting step for the intracellular hydrolysis of S1P. We hypothesized that S1P limits its own degradation by an AMP-kinase (AMPK) dependent modulation of CFTR conductance. Methods: CFTR conductance was assessed using the iodide efflux technique (conventional and real-time) in baby hamster kidney (BHK) cells, which stably express human wild type CFTR. AMPK phosphorylation was determined using Western blots and appropriate antibodies. Results: BHK cells were found to endogenously express Sk1, SPP1 and the S1P₁ and S1P₂ receptors (n=8). S1P (1mM, n=6) significantly reduced iodide conductance by 43% (conventional) and 75% (real-time). The negative effect of S1P on CFTR iodide conductance was enhanced following S1P₂ overexpression but reduced following pretreatment with the S1P₂ blocker JTE013 (5mM, n=6). The inhibitory effect of S1P was associated with AMPK phosphorylation and blocked by the AMPK inhibitor Compound C (80mM, n=5). Accordingly, AMPK activation by AICAR (1 mM, n=5) mimicked the effect of S1P on iodide conductance. Moreover, Western blot analysis demonstrated that acute S1P treatment of BHK-CFTR cells increased the phospho-AMPK/AMPK ratio to 1.47±0.216 (n=3) relative to basal (0.460±0.0874, n=3). Finally, the addition of Compound C, in the presence of S1P, returned the p-AMPK/AMPK ratio to basal levels (n=3). Conclusions: The S1P₂ - and AMPK-dependent modulation of CFTR conductance describes a novel pathway through which S1P could potentially regulate its own degradation.