The effect of direct contact between thymic stromal cells and thymocytes on differentiation markers and functions of the latter was studied. The thymic stromal cells included two epithelial and one fibroblast cell lines, previously described. Murine thymocytes were incubated with confluent monolayers of these cells or their supernatants for 24 hr, using monolayers of non-thymic cells and their supernatants as controls. Then, the thymocytes were tested for changes in expression of several surface antigens [Thy-1, Lyt-1, Lyt-2, L3T4, IL-2-receptor (IL-2R)], spontaneous [3H]thymidine incorporation (STI), lectin-induced proliferative response (PR) and lymphokine (IL-2 and IL-3) production. All three thymic stromal cell lines reduced the expression of Thy-1, Lyt-1 and Lyt-2 significantly. The expression of L3T4 was also reduced in some of the experiments, while IL-2R was not expressed by the thymocytes, neither before nor after the co-culture. The thymic stromal cell lines also increased the spontaneous [3H]thymidine incorporation and lymphokine production by the thymocytes and inhibited their proliferative response to lectins. Under the same experimental conditions, the culture supernatants of the thymic stromal cells and the non-thymic cells did not have any effect on the thymocytes, either when collected and used separately or when used in a co-culture system which allowed thymocyte contact with the medium but not with the stromal cells (Transwell system). These results suggest a specific effect of thymic stromal cells, epithelial as well as fibroblasts, on thymocyte maturation. The effect is mediated by direct cell contact and not by secreted factors.