Previous work in this laboratory demonstrated that rat respiratory airway epithelial cells grown from tissue explants undergo concurrent division and differentiation in culture. In the present studies, this model system has been used to optimize conditions for epithelial growth, with the goal of facilitating the culture of epithelium from other organs and species. Fibroblasts appeared to limit the expansion of epithelial outgrowths; their growth was controlled by using delipidated serum, rather than serum, in the culture media, with addition of adrenergic antagonists. In nonsupplemented media, rat tracheal fibroblasts doubled in number in about 3 d. The doubling time was slowed to more than 9 d by the serum substitution in conjunction with the alpha-antagonist phenoxybenzamine. Rat tracheal epithelial cultures maintained under identical conditions doubled in less than 4 d, so that a growth advantage of threefold was achieved. The combination of conditions also seemed to inhibit growth of fibroblasts from other species. However, even when the problem of fibroblast overgrowth was obviated, the respiratory airway epithelia of the mouse, hamster, and marmoset failed to show self-sustaining growth in culture. The rat epithelium continued to grow for over 6 wk. A number of other organs from the rat, notably esophagus, skin, and salivary gland, showed self-sustaining growth after removal of explants. Although epithelial outgrowths were formed by explants from certain organs of the hamster, only those from the kidney and thyroid continued to grow after removal of the tissue explants. Therefore, growth failure in cultured epithelia, quantitatively defined, is common and frequently unrelated to the problem of fibroblast overgrowth. The rat seems to be the species of choice for studies on the isolated epithelia from most organ sites.