NaCl absorption in the medullary thick ascending limb (THAL) is mediated by the apical Na/K/2Cl cotransporter (NKCC2). Hormones that increase cGMP such as NO and natriuretic peptides decrease NaCl absorption by the THAL. However, the mechanism by which cGMP decreases NaCl absorption in THALs is unknown. We hypothesized that cGMP decreases surface NKCC2 levels in the THAL. We used surface biotinylation to measure surface NKCC2 levels in rat mTHAL suspensions. We first tested the effect of the membrane-permeant cGMP analogue dibutyryl-cGMP. Incubating THALs with db-cGMP for 20 min decreased surface NKCC2 levels in a concentration-dependent manner (basal = 100 %; db-cGMP 100µM = 77 ±7 %; 500µM = 54 ±10 % and 1000µM = 61 ±8 %). A different cGMP analogue (8-Br-cGMP) also decreased surface NKCC2 levels by 25 %, (basal = 100 %; 8-Br-cGMP = 75 ±5 %). Incubation of perfused THALs with db-cGMP decreased apical surface NKCC2 labeling as measured by immunofluorescence and confocal microscopy. cGMP-stimulated phosphodiesterase 2 (PDE2) mediates the effect of NO on NaCl absorption. Thus we examined the role of PDE2 and found that PDE2 inhibitors blocked the effect of db-cGMP on surface NKCC2. Also, a non-stimulatory concentration of db-cAMP blocked cGMP's effect. Finally, db-cGMP inhibited THAL net Cl absorption by 48 ± 4%, and this effect was abolished by PDE2 inhibition. We concluded that cGMP decreases NKCC2 in the apical membrane of THALs and this effect is mediated by PDE2. This is an important mechanism by which cGMP inhibits NaCl absorption by the THAL.