Thiazolidinediones are agonists of peroxisome proliferator-activated receptor gamma (PPARgamma) that can induce fluid retention and weight gain through unclear mechanisms. To test a proposed role for the epithelial sodium channel ENaC in thiazolidinedione-induced fluid retention, we used mice with conditionally inactivated alphaENaC in the collecting duct (Scnn1a(loxloxCre) mice). In control mice, rosiglitazone did not alter plasma aldosterone levels or protein expression of ENaC subunits in the kidney, but did increase body weight, plasma volume, and the fluid content of abdominal fat pads, and decreased hematocrit. Scnn1a(loxloxCre) mice provided functional evidence for blunted Na+ uptake in the collecting duct, but still exhibited rosiglitazone-induced fluid retention. Moreover, treatment with rosiglitazone or pioglitazone did not significantly alter the open probability or number of ENaC channels per patch in isolated, split-open cortical collecting ducts of wild-type mice. Finally, patch-clamp studies in primary mouse inner medullary collecting duct cells did not detect ENaC activity but did detect a nonselective cation channel upregulated by pioglitazone. These data argue against a primary and critical role of ENaC in thiazolidinedione-induced fluid retention.