Mesangial cells derived from isolated glomeruli of rat kidney were grown as homogeneous cell lines in culture. They released, into the culture medium, erythropoietin that had free terminal galactosyl residues and was therefore not active in vivo. The production of erythropoietin by these cells was significantly enhanced by either lowering the PO2 in the incubation atmosphere or by adding cobalt chloride to the culture medium. Therefore, mesangial cells in culture may be considered as an in vitro system in which the regulation of erythropoietin production can be studied under well-defined conditions.