Supernatants of a primary rabbit corneal epithelial cell culture and an established corneal cell line (SIRC) were assayed for their ability to enhance mitogen-induced C3H/HeJ mouse thymocyte proliferation. Significant levels of thymocyte-enhancing activity were detected in supernatants from both primary cultures and SIRC. Maximal levels of activity were found after 48 to 72 hr of culture in serum-free medium with 1 X 10(5) cells/ml. When monolayers of SIRC were disrupted. supernatants of these cultures consistently contained levels of activity higher than those of undisrupted control cultures. When supernatants from SIRC cultures (both serum-free and containing greater than 10% fetal calf serum) were subjected to gel filtration on AcA 54 and Sephacryl S-200, corneal epithelial cell-derived thymocyte-activating factor was eluted as two major peaks, between mol. wt 95,000 and 55,000 and mol. wt. 30,000 and 15,000. These results indicate that corneal epithelial cells, similar to keratinocytes, produce an Interleukin 1-like activity lacking species specificity, which enhances the proliferative capacity of thymocytes. Therefore corneal epithelial cells may interact with the immune system through the production of this cytokine.