We previously showed that the inhalational anesthetic isoflurane protects against renal proximal tubule necrosis via isoflurane-mediated stimulation and translocation of sphingosine kinase-1 (SK1) with subsequent synthesis of sphingosine-1-phosphate (S1P) in renal proximal tubule cells (Kim M, Kim M, Kim N, D'Agati VD, Emala CW Sr, Lee HT. Am J Physiol Renal Physiol 293: F1827-F1835, 2007). We also demonstrated that the anti-necrotic and anti-inflammatory effect of isoflurane is due in part to phosphatidylserine (PS) externalization and subsequent release of transforming growth factor-beta1 (TGF-beta1) (Lee HT, Kim M, Kim J, Kim N, Emala CW. Am J Nephrol 27: 416-424, 2007). In this study, we tested the hypothesis that isoflurane, via TGF-beta1 release, increases caveolae formation in the buoyant fraction of the cell membrane of human renal proximal tubule (HK-2) cells to organize SK1 and S1P signaling. To detect SK1 protein in the caveolae/caveolin fractions, we overexpressed human SK1 in HK-2 cells (SK1-HK-2). SK1-HK-2 cells exposed to isoflurane increased caveolae/caveolin formation in the buoyant membrane fractions which contained key signaling intermediates involved in isoflurane-mediated renal tubule protection, including S1P, SK1, ERK MAPK, and TGF-beta1 receptors. Furthermore, treating SK1-HK-2 cells with recombinant TGF-beta1 or PS liposome mixture increased caveolae formation, mimicking the effects of isoflurane. Conversely, TGF-beta1-neutralizing antibody blocked the increase in caveolae formation induced by isoflurane in SK1-HK-2 cells. The increase in SK1 activity in the caveolae-enriched fractions from isoflurane-treated nonlentivirus-infected HK-2 cells, while smaller in magnitude, was qualitatively similar to that found in the SK1-HK-2 cell line. Finally, isoflurane also increased caveolae formation in the kidneys of TGF-beta1 +/+ mice but not in TGF-beta1 +/- mice (mice with reduced levels of TGF-beta1). Our study demonstrates that isoflurane organizes several key cytoprotective signaling intermediates including TGF-beta1 receptors, SK1 and ERK, within the caveolae fraction of the plasma membrane. Our findings may help to unravel the cellular signaling pathways of volatile anesthetic-mediated renal protection and lead to new therapeutic applications of inhalational anesthetics during the perioperative period.