Brain capillary endothelium in vivo contains high levels of gamma-glutamyltranspeptidase activity. In addition, the presence of this enzyme has been used as a marker of neoplastic cells. Normal rat cerebral endothelial cells in culture exhibit a specific activity for gamma-glutamyltranspeptidase of 2 units/10(6) cells. In vitro transformation of these cells is achieved by the use of an avian retrovirus, Schmidt-Ruppin RSV-strain D. The resultant cell line, designated RCE-T1, demonstrates a significant increase in gamma-glutamyltranspeptidase activity up to 20 units/10(6) cells in early passage levels (9-26) after which enzyme activity declines and returns to normal levels by passage 80. This variation in enzyme activity correlates with histochemical staining for this enzyme. Furthermore, the enzyme activity increases linearly over a 1000-fold range of cell concentrations. Various culture modifications do not influence this pattern of enzyme expression. These parameters include trypsin dissociation, cell freezing, degree of confluency and culture maintenance with serum or with conditioned medium obtained from passage levels exhibiting high or low enzyme activity. RCE-T1 cells will provide a unique model system to study the distribution and regulation of this enzyme during differentiation and viral carcinogenesis.