Transforming growth factor-β regulates tubular epithelial-myofibroblast transdifferentiation in vitro. Background. We recently found evidence of tubular epithe-lial-myofibroblast transdifferentiation (TEMT) during the de-velopment of tubulointerstitial fibrosis in the rat remnant kid-ney. This study investigated the mechanisms that induce TEMT in vitro. Methods. The normal rat kidney tubular epithelial cell line (NRK52E) was cultured for six days on plastic or collagen type I-coated plates in the presence or absence of recombinant trans-forming growth factor-j31 (TGF-β1). Transdifferentiation of tubular cells into myofibroblasts was assessed by electron mi-croscopy and by expression of α-smooth muscle actin (α-SMA) and E-cadherin. Results. NRK52E cells cultured on plastic or collagen-coated plates showed a classic cobblestone morphology. Culture in 1 ng/ml TGF-β caused only very minor changes in morphology, but culture in 10 or 50ng/ml TGF-β1 caused profound changes. This involved hypertrophy, a loss of apical-basal polarity and microvilli, with cells becoming elongated and invasive, the for-mation of a new front-end back-end polarity, and the appear-ance of actin microfilaments and dense bodies. These morpho-logjcal changes were accompanied by phenotypic changes. Double immunohistochemistry staining showed that the addi-tion of TGF-β1 to confluent cell cultures caused a loss of the epithelial marker E-cadherin and de novo expression of α-SMA. An intermediate stage in transdifferentiation could be seen with hypertrophic cells expressing both E-cadherin and α-SMA. De novo α-SMA expression was confirmed by Northern blotting, Western blotting, and flow cytometry. In particular, cells with a transformed morphology showed strong α-SMA immunostaining of characteristic microfilament struc-tures along the cell axis. There was a dose-dependent increase in the percentage of cells expressing α-SMA with increasing concentrations of TGF-β1, which was completely inhibited by the addition of a neutrafizing ant-TGF-β1 antibody. Corn