A new medium composition was established for growth of adult human corneal endothelial cells (HCEC) under serum-reduced conditions. Growth assays were performed in clonal densities with HCEC using serum-reduced culture conditions. The growth-promoting effect of different substances was tested step by step. The new serum-reduced medium was compared with the generally used medium supplemented with conventional amounts of serum. A culture medium recently described (Engelmann K, Böhnke M, Friedl P. Optimization of culture conditions for human corneal endothelial cells. In Vitro Cell Dev Biol 1989;25:1065-72) for the long-term cultivation of human corneal endothelial cells derived from adult donors was used as the starting point for the development of a new low-serum medium. It was found that the addition to the supplements ascorbic acid, insulin, selenium, transferrin, lipids, and fibroblast growth factor to the basal medium F99 (a 1:1 mixture of Ham's F12 and M199) allowed reduction of the serum content to as low as 2%. The new medium formula, called F99sr, showed an improved dose-response curve of cell growth to serum content over the range 2-40%. To ensure the maintenance of a typical endothelial cell morphology at confluence, the serum content should be 5%.