The in vitro culture of human kidney epithelial cells of defined nephronal origin would prove valuable in a variety of studies defining the factors and mechanisms responsible for diseases and disorders of the kidney. In this study we have tested the hypothesis that employing a serum-free growth medium allows the selective cultivation of human kidney epithelial cells. It is demonstrated that human kidney cortex, explanted into serum-free hormonally defined growth medium gives rise to a primary culture of kidney epithelial cells of homogeneous morphology capable of hemicyst formation. While these cells were able to proliferate to confluency as an explant culture, they were unable to undergo stable subculture. Subsequent manipulation of the culture vessel surface (an initial coat of bovine type I collagen followed by the absorption of macromolecules from fetal calf serum) yielded cultures able to be subcultured with growth to at least 30 cell generations. These cells were identified to be of proximal tubule origin by employment of enzyme histochemistry, immunohistochemistry, and ultrastructural examination.